Novel plant expression constructs

ABSTRACT

The present invention relates to novel plant expression constructs. More specifically the present invention provides DNA constructs comprising 5′ regulatory sequences for modulating the expression of operably linked genes in plants.

FIELD OF THE INVENTION

[0001] The present invention relates to the isolation and use of nucleicacid molecules for control of gene expression in plants, specificallynovel plant promoters.

BACKGROUND OF THE INVENTION

[0002] One of the goals of plant genetic engineering is to produceplants with agronomically important characteristics or traits. Recentadvances in genetic engineering have provided the requisite tools toproduce transgenic plants that contain and express foreign genes (Kahlet al., World J. of Microbiol. Biotech. 11:449-460, 1995). Particularlydesirable traits or qualities of interest for plant genetic engineeringwould include but are not limited to resistance to insects, fungaldiseases, and other pests and disease-causing agents, tolerances toherbicides, enhanced stability or shelf-life, yield, environmentaltolerances, and nutritional enhancements. The technological advances inplant transformation and regeneration have enabled researchers to takeexogenous DNA, such as a gene or genes from a heterologous or a nativesource, and incorporate the exogenous DNA into the plant's genome. Inone approach, expression of a novel gene that is not normally expressedin a particular plant or plant tissue may confer a desired phenotypiceffect. In another approach, transcription of a gene or part of a genein an antisense orientation may produce a desirable effect by preventingor inhibiting expression of an endogenous gene.

[0003] In order to produce a transgenic plant, a construct that includesa heterologous gene sequence that confers a desired phenotype whenexpressed in the plant is introduced into a plant cell. The constructalso includes a plant promoter that is operably linked to theheterologous gene sequence, often a promoter not normally associatedwith the heterologous gene. The construct is then introduced into aplant cell to produce a transformed plant cell, and the transformedplant cell is regenerated into a transgenic plant. The promoter controlsexpression of the introduced DNA sequence to which the promoter isoperably linked and thus affects the desired characteristic conferred bythe DNA sequence.

[0004] It would be advantageous to have a variety of promoters to tailorgene expression such that a gene or gene(s) is transcribed efficientlyat the right time during plant growth and development, in the optimallocation in the plant, and in the amount necessary to produce thedesired effect. For example, constitutive expression of a gene productmay be beneficial in one location of the plant but less beneficial inanother part of the plant. In other cases, it may be beneficial to havea gene product produced at a certain developmental stage of the plant orin response to certain environmental or chemical stimuli. The commercialdevelopment of genetically improved germplasm has also advanced to thestage of introducing multiple traits into crop plants, often referred toas a gene stacking approach. In this approach, multiple genes conferringdifferent characteristics of interest can be introduced into a plant. Itis important when introducing multiple genes into a plant that each geneis modulated or controlled for optimal expression and that theregulatory elements are diverse in order to reduce the potential of genesilencing. In light of these and other considerations, it is apparentthat optimal control of gene expression and regulatory element diversityare important in plant biotechnology.

SUMMARY OF THE INVENTION

[0005] The present invention relates to DNA plant expression constructsthat comprise Arabidopsis actin (Act) promoter sequences Act1a, Act1b,Act 2, Act3, Act7, Act8, Act11, Act12 and the elongation factor 1α(EF1α) promoter sequence, and fragments and cis elements derived fromthese promoters operably linked to heterologous structural genesequences that function in crop plant cells.

[0006] Thus, according to one embodiment of the invention, a recombinantDNA construct is provided that comprises, in operable linkage, apromoter that is functional in a cell of a crop plant, the promotercomprising: at least one cis element derived from SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, and SEQ ID NO:26; a structural DNA sequenceheterologous to the promoter; and a 3′ non-translated region thatfunctions in plants to cause the addition of polyadenylated nucleotidesto the 3′ end of the RNA sequence. For example, the promoter may consistessentially of a 5′ regulatory region derived from any of SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26 (including or excluding anyintron sequences located therein). The structural gene may comprise anyheterologous nucleotide sequence wherein expression of the sequenceresults in an agronomically useful trait or product in a transgenic cropplant.

[0007] According to another aspect of the invention is a DNA constructcomprising a structural DNA sequence operably linked to the promotersequences of the present invention that encode a protein employed toconfer herbicide tolerance to a crop plant. This herbicide toleranceprotein includes, but is not limited to glyphosate tolerance proteingenes such as a glyphosate resistant EPSP synthase gene alone, or incombination with one or more glyphosate degrading protein genes.

[0008] According to another embodiment of the invention, DNA constructssuch as those described above are provided wherein the promoter is ahybrid or chimeric promoter comprising at least one cis element derivedfrom one or more of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQID NO:26 operably linked to a heterologous promoter sequence such as acaulimovirus promoter, for example the Cauliflower mosaic virus 35Spromoter or the Figwort mosaic virus promoter.

[0009] According to another embodiment of the invention, DNA constructs,such as those described above, are provided in tandem, wherein thepromoter is a hybrid or chimeric promoter comprising at least one ciselement derived from one or more of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, and SEQ ID NO:26 operably linked to a heterologous gene sequencethat expresses in transgenic crop plant cells. The chimeric promotersequences more specifically comprising the sequences identified in SEQID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0010] According to another embodiment of the invention, a DNA constructsuch as that described above is provided wherein the structural DNAsequence is a glyphosate tolerance gene, such that when the DNAconstruct is introduced into a plant cell, it confers to the plant celltolerance to an aqueous glyphosate formulation that includes at least 50grams acid equivalent per liter (g a.e./1) of glyphosate. In otherrelated embodiments, the DNA construct confers to the plant celltolerance to glyphosate formulations having higher glyphosateconcentrations (for example, at least 300 grams acid equivalent perliter of glyphosate. According to one embodiment, the DNA constructconfers to the plant cell tolerance to at least one application ofRoundup Ultra® at a rate of 16 ounces (oz) per acre, for example, and inother embodiments, glyphosate tolerance extends to one to two or moreapplications of 16 oz per acre, 32 oz per acre, or 64 oz per acre, forexample.

[0011] According to another embodiment of the invention, transgenic cropplants are provided that are transformed with a DNA construct asdescribed above, including monocot species and dicot species. We havediscovered that the Arabidopsis actin and Arabidopsis EF1α promoters aresufficiently active in other crop plant species such as cotton, tomato,and sunflower, for example, that when used to control expression of aglyphosate tolerance gene, such as aroA:CP4, the plants toleratecommercial application rates of glyphosate, exhibiting good vegetativetolerance and low damage to reproductive tissues. Such promoters canalso be used to express other genes of interest in plants, including,but not limited to, genes that confer herbicide tolerance, insectcontrol, disease resistance, increased stability or shelf, higher yield,nutritional enhancement, expression of a pharmaceutical or other desiredpolypeptide product, or a desirable change in plant physiology ormorphology, and so on.

[0012] According to another embodiment of the invention, transgenic cropplants are provided that are transformed with multiple DNA constructscomprising the Arabidopsis actin and Arabidopsis EF1α promoters aresufficiently active in other plant species such as cotton, tomato,sunflower, for example, that when used to control expression of aglyphosate tolerance gene such as aroA:CP4, the plants toleratedcommercial application rates of glyphosate, exhibiting good vegetativetolerance and low damage to reproductive tissues. Such promoters canalso be used to express other genes of interest in plants, including,but not limited to, genes that confer herbicide tolerance, insectcontrol, disease resistance, increased stability or shelf, higher yield,nutritional enhancement, expression of a pharmaceutical or other desiredpolypeptide product, or a desirable change in plant physiology ormorphology, and so on.

[0013] According to another embodiment of the invention, transgenic cropplants are provided that are transformed with DNA constructs comprisingthe Arabidopsis actin and Arabidopsis EF1α promoters as chimeric DNAmolecules in fusion with caulimovirus DNA molecules having promoteractivity in plants sufficiently active in other plant species such ascotton, tomato, canola, soybean, and sunflower, for example, that whenused to control expression of a glyphosate tolerance gene such asaroA:CP4, the plants tolerate commercial application rates ofglyphosate, exhibiting good vegetative tolerance and low damage toreproductive tissues. Such promoters can also be used to express othergenes of interest in plants, including, but not limited to, genes thatconfer herbicide tolerance, insect control, disease resistance,increased stability or shelf, higher yield, nutritional enhancement,expression of a pharmaceutical or other desired polypeptide product, ora desirable change in plant physiology or morphology, and so on.

[0014] According to another embodiment of the invention methods areprovided for expressing a structural DNA sequence in a plant. Suchmethods comprise, providing a DNA construct as described above,introducing the DNA construct into a plant cell, and regenerating theplant cell to produce a plant such that the structural DNA isexpressible in the plant. According to a related embodiment, a method ofcontrolling weeds is provided in which the DNA construct comprises aglyphosate tolerance gene and one applies to a crop plant transformedwith the DNA construct an amount of glyphosate that is sufficient tocontrol weeds without significantly damaging the crop plant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a plasmid map of pCGN8086

[0016]FIG. 2 is a plasmid map of pMON45325

[0017]FIG. 3 is a plasmid map of pMON45331

[0018]FIG. 4 is a plasmid map of pMON45332

[0019]FIG. 5 is a plasmid map of pCGN9190

[0020]FIG. 6 is a plasmid map of pCGN9153

[0021]FIG. 7 is a plasmid map of pCGN8099

[0022]FIG. 8 is a plasmid map of pCGN8088

[0023]FIG. 9 is a plasmid map of pCGN8068

[0024]FIG. 10 is a plasmid map of pCGN8096

[0025]FIG. 11 is a plasmid map of pCGN9151

[0026]FIG. 12 is a plasmid map of pMON10156

[0027]FIG. 13 is a plasmid map of pMON52059

[0028]FIG. 14 is a plasmid map of pMON54952

[0029]FIG. 15 is a plasmid map of pMON54953

[0030]FIG. 16 is a plasmid map of pMON54954

[0031]FIG. 17 is a plasmid map of pMON54955

[0032]FIG. 18 is a plasmid map of pMON54956

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0033] SEQ ID NO:1 is the forward PCR primer used for the isolation ofthe Act2 promoter

[0034] SEQ ID NO:2 is the reverse PCR primer used for the isolation ofthe Act2 promoter

[0035] SEQ ID NO:3 is the forward PCR primer used for the isolation ofthe Act8 promoter

[0036] SEQ ID NO:4 is the reverse PCR primer used for the isolation ofthe Act8 promoter

[0037] SEQ ID NO:5 is the forward PCR primer used for the isolation ofthe Act11 promoter

[0038] SEQ ID NO:6 is the reverse PCR primer used for the isolation ofthe Act11 promoter

[0039] SEQ ID NO:7 is the forward PCR primer used for the isolation ofthe EF1 promoter

[0040] SEQ ID NO:8 is the reverse PCR primer used for the isolation ofthe EF1 promoter

[0041] SEQ ID NO:9 is the sequence of the Act2 promoter including theintron sequence of the Act2 gene. Base positions 1-764 represent thepromoter sequence; base positions 765-1215 represent the intron followedby 5 bases of 5′ untranslated region (5′ UTR) prior to the ATG; thetranscription start site is located at base position 597.

[0042] SEQ ID NO:10 is the sequence of the Act8 promoter including thefirst intron of the Act8 gene. Base positions 1-797 represent thepromoter sequence; base positions 798-1259 represent the intron followedby 10 bases of 5′ UTR prior to the ATG; the transcription start site islocated at base position 646.

[0043] SEQ ID NO:11 is the sequence of the Act11 promoter including thefirst intron of the Act11 gene. Base positions 1-1218 represent thepromoter sequence; base positions 1219-1381 represent the intronfollowed by 10 bases of 5′ UTR prior to the ATG; the transcription startsite is located at base position 1062.

[0044] SEQ ID NO:12 is the sequence of the EF1 promoter including thefirst intron of the EF1 gene. Base positions 1-536 represent thepromoter sequence; base positions 537-1137 represent the intron followedby 22 bases of 5′ UTR prior to the ATG; the transcription start site islocated at base position 481.

[0045] SEQ ID NO:13 is the forward PCR primer used for the isolation ofthe Act1a promoter

[0046] SEQ ID NO:14 is the forward PCR primer used for the isolation ofthe Act1b promoter

[0047] SEQ ID NO:15 is the reverse PCR primer used for the isolation ofthe Act1a and Act1b promoter

[0048] SEQ ID NO:16 is the forward PCR primer used for the isolation ofthe Act3 promoter

[0049] SEQ ID NO:17 is the reverse PCR primer used for the isolation ofthe Act3 promoter

[0050] SEQ ID NO:18 is the forward PCR primer used for the isolation ofthe Act7 promoter

[0051] SEQ ID NO:19 is the reverse PCR primer used for the isolation ofthe Act7 promoter

[0052] SEQ ID NO:20 is the forward PCR primer used for the isolation ofthe Act12 promoter

[0053] SEQ ID NO:21 is the reverse PCR primer used for the isolation ofthe Act12 promoter

[0054] SEQ ID NO:22 is the sequence of the Act1a promoter including thefirst intron of the Act1a gene. Base positions 1-1033 represent thepromoter sequence; base positions 1034-1578 represent the intron and 5′UTR.

[0055] SEQ ID NO:23 is the sequence of the Act1b promoter including thefirst intron of the Act1b gene. Base positions 1-914 represent thepromoter sequence; base positions 915-1468 represent the intron and 5′UTR sequence.

[0056] SEQ ID NO:24 is the sequence of the Act3 promoter including thefirst intron of the Act3 gene. Base positions 1-1023 represent thepromoter sequence; base positions 1024-1642 represent the intron and 5′UTR sequence.

[0057] SEQ ID NO:25 is the sequence of the Act7 promoter including thefirst intron of the Act7 gene. Base positions 1-600 represent thepromoter sequence; base positions 601-1241 represent the intron and 5′UTR sequence.

[0058] SEQ ID NO:26 is the sequence of the Act 12 promoter including thefirst intron of the Act12 gene. Base positions 1-1017 represent thepromoter sequence; base positions 1018-1313 represent the intron and 5′UTR sequence.

[0059] SEQ ID NO:27 is the sequence of the chimeric FMV-Act11 promoterincluding the first intron of the Act11 gene. Base positions 1-536represent the duplicated FMV promoter sequence; base positions 553-1946represent the Arabidopsis Actin 11 promoter, intron and 5′ UTR sequence.

[0060] SEQ ID NO:28 is the sequence of the chimeric FMV-EF1α promoterincluding the first intron of the EF1α gene. Base positions 1-536present the FMV promoter sequence; base positions 553-1695 represent theEF1α promoter, intron and 5′ UTR sequence.

[0061] SEQ ID NO:29 is the sequence of the CaMV-Act8 promoter includingthe first intron of the Act8 gene. Base positions 1-523 present the CaMVpromoter sequence; base positions 534-1800 represent the Act8 promoter,intron and 5′ UTR sequence.

[0062] SEQ ID NO:30 is the sequence of the CaMV-Act2 promoter includingthe first intron of the Act2 gene. Base positions 1-523 represent theCaMV promoter sequence; base positions 534-1742 represent the Act2promoter, intron and 5′ UTR sequence.

DETAILED DESCRIPTION OF THE INVENTION

[0063] This application claims the benefit of U.S. ProvisionalApplication No. 60/171,173, filed Dec. 16, 1999. The followingdefinitions and methods are provided to better define, and to guidethose of ordinary skill in the art in the practice of, the presentinvention. Unless otherwise noted, terms are to be understood accordingto conventional usage by those of ordinary skill in the relevant art.The nomenclature for DNA bases as set forth at 37 CFR § 1.822 is used.The standard one- and three-letter nomenclature for amino acid residuesis used. “Nucleic acid (sequence)” or “polynucleotide (sequence)” refersto single- or double-stranded DNA or RNA of genomic or synthetic origin,i.e., a polymer of deoxyribonucleotide or ribonucleotide bases,respectively, read from the 5′ (upstream) end to the 3′ (downstream)end. The nucleic acid can represent the sense or complementary(antisense) strand.

[0064] “Native” refers to a naturally occurring (“wild-type”) nucleicacid sequence.

[0065] “Heterologous” sequence refers to a sequence which originatesfrom a foreign source or species or, if from the same source, ismodified from its original form.

[0066] An “isolated” nucleic acid sequence is substantially separated orpurified away from other nucleic acid sequences with which the nucleicacid is normally associated in the cell of the organism in which thenucleic acid naturally occurs, i.e., other chromosomal orextrachromosomal DNA. The term embraces nucleic acids that arebiochemically purified so as to substantially remove contaminatingnucleic acids and other cellular components. The term also embracesrecombinant nucleic acids and chemically synthesized nucleic acids. Theterm “substantially purified”, as used herein, refers to a moleculeseparated from other molecules normally associated with it in its nativestate. More preferably, a substantially purified molecule is thepredominant species present in a preparation. A substantially purifiedmolecule may be greater than 60% free, preferably 75% free, morepreferably 90% free from the other molecules (exclusive of solvent)present in the natural mixture. The term “substantially purified” is notintended to encompass molecules present in their native state.

[0067] A first nucleic acid sequence displays “substantially identity”to a reference nucleic acid sequence if, when optimally aligned (withappropriate nucleotide insertions or deletions totaling less than 20percent of the reference sequence over the window of comparison) withthe other nucleic acid (or its complementary strand), there is at leastabout 75% nucleotide sequence identity, preferably at least about 80%identity, more preferably at least about 85% identity, and mostpreferably at least about 90% identity over a comparison window of atleast 20 nucleotide positions, preferably at least 50 nucleotidepositions, more preferably at least 100 nucleotide positions, and mostpreferably over the entire length of the first nucleic acid. Optimalalignment of sequences for aligning a comparison window may be conductedby the local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482, 1981; by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443, 1970; by the search for similarity methodof Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988;preferably by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA) in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis. Thereference nucleic acid may be a full-length molecule or a portion of alonger molecule. Alternatively, two nucleic acids are have substantialidentity if one hybridizes to the other under stringent conditions, asdefined below.

[0068] A first nucleic acid sequence is “operably linked” with a secondnucleic acid sequence when the sequences are so arranged that the firstnucleic acid sequence affects the function of the second nucleic-acidsequence. Preferably, the two sequences are part of a single contiguousnucleic acid molecule and more preferably are adjacent. For example, apromoter is operably linked to a gene if the promoter regulates ormediates transcription of the gene in a cell.

[0069] A “recombinant” nucleic acid is made by an artificial combinationof two otherwise separated segments of sequence, e.g., by chemicalsynthesis or by the manipulation of isolated segments of nucleic acidsby genetic engineering techniques. Techniques for nucleic-acidmanipulation are well-known (see for example Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, 1989; Mailga etal., Methods in Plant Molecular Biology, Cold Spring Harbor Press, 1995;Birren et al., Genome Analysis: volume 1, Analyzing DNA, (1997), volume2, Detecting Genes, (1998), volume 3, Cloning Systems, (1999) volume 4,Mapping Genomes, (1999), Cold Spring Harbor, N.Y.).

[0070] Methods for chemical synthesis of nucleic acids are discussed,for example, in Beaucage and Carruthers, Tetra. Letts. 22:1859-1862,1981, and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemicalsynthesis of nucleic acids can be performed, for example, on commercialautomated oligonucleotide synthesizers.

[0071] A “synthetic nucleic acid sequence” can be designed andchemically synthesized for enhanced expression in particular host cellsand for the purposes of cloning into appropriate constructs. Host cellsoften display a preferred pattern of codon usage (Murray et al., 1989).Synthetic DNAs designed to enhance expression in a particular hostshould therefore reflect the pattern of codon usage in the host cell.Computer programs are available for these purposes including but notlimited to the “BestFit” or “Gap” programs of the Sequence AnalysisSoftware Package, Genetics Computer Group, Inc., University of WisconsinBiotechnology Center, Madison, Wis. 53711.

[0072] “Amplification” of nucleic acids or “nucleic acid reproduction ”refers to the production of additional copies of a nucleic acid sequenceand is carried out using polymerase chain reaction (PCR) technologies. Avariety of amplification methods are known in the art and are described,inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in PCRProtocols: A Guide to Methods and Applications, ed. Innis et al.,Academic Press, San Diego, 1990. In PCR, a primer refers to a shortoligonucleotide of defined sequence which is annealed to a DNA templateto initiate the polymerase chain reaction.

[0073] “Transformed”, “transfected”, or “transgenic” refers to a cell,tissue, organ, or organism into which has been introduced a foreignnucleic acid, such as a recombinant construct. Preferably, theintroduced nucleic acid is integrated into the genomic DNA of therecipient cell, tissue, organ or organism such that the introducednucleic acid is inherited by subsequent progeny. A “transgenic” or“transformed” cell or organism also includes progeny of the cell ororganism and progeny produced from a breeding program employing such a“transgenic” plant as a parent in a cross and exhibiting an alteredphenotype resulting from the presence of a recombinant construct orconstruct.

[0074] The term “gene” refers to chromosomal DNA, plasmid DNA, cDNA,synthetic DNA, or other DNA that encodes a peptide, polypeptide,protein, or RNA molecule, and regions flanking the coding sequenceinvolved in the regulation of expression. Some genes can be transcribedinto mRNA and translated into polypeptides (structural genes); othergenes can be transcribed into RNA (e.g. rRNA, tRNA); and other types ofgene function as regulators of expression (regulator genes).

[0075] “Expression” of a gene refers to the transcription of a gene toproduce the corresponding MRNA and translation of this mRNA to producethe corresponding gene product, i.e., a peptide, polypeptide, orprotein. Gene expression is controlled or modulated by regulatoryelements including 5′ regulatory elements such as promoters.

[0076] “Genetic component” refers to any nucleic acid sequence orgenetic element which may also be a component or part of an expressionconstruct. Examples of genetic components include, but are not limitedto promoter regions, 5′ untranslated leaders, introns, genes, 3′untranslated regions, and other regulatory sequences or sequences whichaffect transcription or translation of one or more nucleic acidsequences.

[0077] The terms “recombinant DNA construct”, “recombinant construct”,“expression construct” or “expression cassette” refer to any agent suchas a plasmid, cosmid, virus, BAC (bacterial artificial chromosome),autonomously replicating sequence, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA nucleotide sequence,derived from any source, capable of genomic integration or autonomousreplication, comprising a DNA molecule in which one or more DNAsequences have been linked in a functionally operative manner usingwell-known recombinant DNA techniques.

[0078] “Complementary” refers to the natural association of nucleic acidsequences by base-pairing (A-G-T pairs with the complementary sequenceT-C-A). Complementarity between two single-stranded molecules may bepartial, if only some of the nucleic acids pair are complementary; orcomplete, if all bases pair are complementary. The degree ofcomplementarity affects the efficiency and strength of hybridization andamplification reactions.

[0079] “Homology” refers to the level of similarity between nucleic acidor amino acid sequences in terms of percent nucleotide or amino acidpositional identity, respectively, i.e., sequence similarity oridentity. Homology also refers to the concept of similar functionalproperties among different nucleic acids or proteins.

[0080] “Promoter” refers to a nucleic acid sequence located upstream or5′ to a translational start codon of an open reading frame (orprotein-coding region) of a gene and that is involved in recognition andbinding of RNA polymerase II and other proteins (trans-actingtranscription factors) to initiate transcription. A “plant promoter” isa native or non-native promoter that is functional in plant cells.Constitutive promoters are functional in most or all tissues of a plantthroughout plant development. Tissue-, organ- or cell-specific promotersare expressed only or predominantly in a particular tissue, organ, orcell type, respectively. Rather than being expressed “specifically” in agiven tissue, organ, or cell type, a promoter may display “enhanced”expression, i.e., a higher level of expression, in one part (e.g., celltype, tissue, or organ) of the plant compared to other parts of theplant. Temporally regulated promoters are functional only orpredominantly during certain periods of plant development or at certaintimes of day, as in the case of genes associated with circadian rhythm,for example. Inducible promoters selectively express an operably linkedDNA sequence in response to the presence of an endogenous or exogenousstimulus, for example by chemical compounds (chemical inducers) or inresponse to environmental, hormonal, chemical, and/or developmentalsignals. Inducible or regulated promoters include, for example,promoters regulated by light, heat, stress, flooding or drought,phytohormones, wounding, or chemicals such as ethanol, jasmonate,salicylic acid, or safeners.

[0081] Any plant promoter can be used as a 5′ regulatory sequence formodulating expression of a particular gene or genes. One preferredpromoter would be a plant RNA polymerase II promoter. Plant RNApolymerase II promoters, like those of other higher eukaryotes, havecomplex structures and are comprised of several distinct elements. Onesuch element is the TATA box or Goldberg-Hogness box, which is requiredfor correct expression of eukaryotic genes in vitro and accurate,efficient initiation of transcription in vivo. The TATA box is typicallypositioned at approximately −25 to −35, that is, at 25 to 35 basepairs(bp) upstream (5′) of the transcription initiation site, or cap site,which is defined as position +1 (Breathnach and Chambon, Ann. Rev.Biochem. 50:349-383, 1981; Messing et al., In: Genetic Engineering ofPlants, Kosuge et al., eds., pp. 211-227, 1983). Another common element,the CCAAT box, is located between −70 and −100 bp. In plants, the CCAATbox may have a different consensus sequence than the functionallyanalogous sequence of mammalian promoters (the plant analogue has beentermed the “AGGA box” to differentiate it from its animal counterpart;Messing et al., In: Genetic Engineering of Plants, Kosuge et al., eds.,pp. 211-227, 1983). In addition, virtually all promoters includeadditional upstream activating sequences or enhancers (Benoist andChambon, Nature 290:304-310, 1981; Gruss et al., Proc. Nat. Acad. Sci.USA 78:943-947, 1981; and Khoury and Gruss, Cell 27:313-314, 1983)extending from around −100 bp to −1,000 bp or more upstream of thetranscription initiation site.

[0082] When fused to heterologous DNA sequences, such promoterstypically cause the fused sequence to be transcribed in a manner that issimilar to that of the gene sequence with which the promoter is normallyassociated. Promoter fragments that include regulatory sequences can beadded (for example, fused to the 5′ end of, or inserted within, anactive promoter having its own partial or complete regulatory sequences(Fluhr et al., Science 232:1106-1112, 1986; Ellis et al., EMBO J.6:11-16, 1987; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986-8990, 1987; Poulsen and Chua, Mol. Gen. Genet. 214:16-23, 1988;Comai et al., Plant Mol. Biol. 15:373-381, 1991). Alternatively,heterologous regulatory sequences can be added to the 5′ upstream regionof an inactive, truncated promoter, e.g., a promoter including only thecore TATA and, sometimes, the CCAAT elements (Fluhr et al., Science232:1106-1112, 1986; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986-8990, 1987; Aryan et al., Mol. Gen. Genet. 225:65-71, 1991).

[0083] Promoters are typically comprised of multiple distinct“cis-acting transcriptional regulatory elements,” or simply“cis-elements,” each of which confers a different aspect of the overallcontrol of gene expression (Strittmatter and Chua, Proc. Nat. Acad. Sci.USA 84:8986-8990, 1987; Ellis et al., EMBO J. 6:11-16, 1987; Benfey etal., EMBO J. 9:1677-1684, 1990). “Cis elements” bind trans-actingprotein factors that regulate transcription. Some cis elements bind morethan one factor, and trans-acting transcription factors may interactwith different affinities with more than one cis element (Johnson andMcKnight, Ann. Rev. Biochem. 58:799-839, 1989). Plant transcriptionfactors, corresponding cis elements, and analysis of their interactionare discussed, for example, In: Martin, Curr. Opinions Biotech.7:130-138, 1996; Murai, In: Methods in Plant Biochemistry and MolecularBiology, Dashek, ed., CRC Press, 1997, pp. 397-422; and Methods in PlantMolecular Biology, Maliga et al., eds., Cold Spring Harbor Press, 1995,pp. 233-300. The promoter sequences of the present invention can contain“cis elements” that confer or modulate gene expression.

[0084] Cis elements can be identified by a number of techniques,including deletion analysis, i.e., deleting one or more nucleotides fromthe 5′ end or internal to a promoter; DNA binding protein analysis usingDnase I footprinting, methylation interference, electrophoresismobility-shift assays, in. vivo genomic footprinting byligation-mediated PCR and other conventional assays; or by sequencesimilarity with known cis element motifs by conventional sequencecomparison methods. The fine structure of a cis element can be furtherstudies by mutagenesis (or substitution) of one or more nucleotides ofthe element or by other conventional methods (see for example, Methodsin Plant Biochemistry and Molecular Biology, Dashek, ed., CRC Press,1997, pp. 397-422; and Methods in Plant Molecular Biology, Maliga etal., eds., Cold Spring Harbor Press, 1995, pp. 233-300).

[0085] Cis elements can be obtained by chemical synthesis or by cloningfrom promoters that include such elements. Cis elements can also besynthesized with additional flanking sequences that contain usefulrestriction enzyme sites to facilitate subsequence manipulation. In oneembodiment, the promoters are comprised of multiple distinct“cis-elements”. In a preferred embodiment sequence regions comprising“cis elements” of the nucleic acid sequences of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, and SEQ ID NO:26 are identified using computerprograms including, but not limited to MEME or SIGNALSCAN that aredesigned specifically to identify cis elements, or domains or motifswithin sequences.

[0086] The present invention includes fragments or cis elements of SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26 or homologues of ciselements known to effect gene regulation that show homology with thenucleic acid sequences of the present invention. Such nucleic acidfragments can include any region of the disclosed sequences. Thepromoter regions or partial promoter regions of the present invention asshown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26 cancontain at least one regulatory element including, but not limited to“cis elements” or domains that are capable of regulating expression ofoperably linked DNA sequences, such as in male reproductive tissues.

[0087] Plant promoters can also include promoters produced through themanipulation of known promoters to produce synthetic, chimeric, orhybrid promoters. Such promoters can also combine cis elements from oneor more promoters, for example, by adding a heterologous regulatorysequence to an active promoter with its own partial or completeregulatory sequences (Ellis et al., EMBO J. 6:11-16, 1987; Strittmatterand Chua, Proc. Nat. Acad. Sci. USA 84:8986-8990, 1987; Poulsen andChua, Mol. Gen. Genet. 214:16-23, 1988; Comai et al., Plant. Mol. Biol.15:373-381, 1991). Chimeric promoters have also been developed by addinga heterologous regulatory sequence to the 5′ upstream region of aninactive, truncated promoter, i.e., a promoter that includes only thecore TATA and, optionally, the CCAAT elements (Fluhr et al., Science232:1106-1112, 1986; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986-8990, 1987; Aryan et al., Mol. Gen. Genet. 225:65-71, 1991).

[0088] Chimeric or hybrid promoters according to the present inventionmay include at least one known cis element such as elements that areregulated by numerous environmental factors such as light, heat, orstress; elements that are regulated or induced by pathogens orchemicals, and the like. Such elements may either positively ornegatively regulate gene expression, depending on the conditions.Examples of cis elements include, but are not limited to oxygenresponsive elements (Cowen et al., J. Biol. Chem. 268(36):26904, 1993),light regulatory elements (see for example, Bruce and Quail, Plant Cell2: 1081, 1990, and Bruce et al., EMBO J. 10:3015, 1991, a cis elementreponsive to methyl jasmonate treatment (Beaudoin and Rothstein, PlantMol. Biol. 33:835, 1997, salicylic acid-responsive elements (Strange etal., Plant J. 11:1315, 1997, heat shock response elements (Pelham etal., Trends Genet. 1:31, 1985, elements responsive to wounding andabiotic stress (Loace et al., Proc. Natl. Acad. Sci. U.S.A. 89:9230,1992; Mhiri et al., Plant Mol. Biol. 33:257, 1997), cold-responsiveelements (Baker et al., Plant Mol. Biol. 24:701, 1994; Jiang et al.,Plant Mol. Biol. 30:679, 1996; Nordin et al., Plant Mol. Biol. 21:641,1993; Zhou et al., J. Biol. Chem. 267:23515, 1992), and droughtresponsive elements, (Yamaguchi et al., Plant Cell 6:251-264, 1994; Wanget al., Plant Mol. Biol. 28:605, 1995; Bray E. A. Trends in PlantScience 2:48, 1997).

[0089] In another embodiment, the nucleotide sequences as shown in SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NOS:27, SEQ IDNO:28, SEQ ID NO:29, and SEQ ID NO:30 includes any length of saidnucleotide sequences that is capable of regulating an operably linkedDNA sequence. For example, the sequences as disclosed in SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NOS:27, SEQ ID NO:28,SEQ ID NO:29, and SEQ ID NO:30 may be truncated or have portions deletedand still be capable of regulating transcription of an operably linkedDNA sequence. In a related embodiment, a cis element of the disclosedsequences may confer a particular specificity such as conferringenhanced expression of operably linked DNA sequences in certain tissues.Consequently, any sequence fragments, portions, or regions of thedisclosed sequences of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NOS:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 canbe used as regulatory sequences including but not limited to ciselements or motifs of the disclosed sequences. For example, one or morebase pairs may be deleted from the 5′ or 3′ end of a promoter sequenceto produce a “truncated” promoter. One or more base pairs can also beinserted, deleted, or substituted internally to a promoter sequence.Promoters can be constructed such that promoter fragments or elementsare operably linked, for example, by placing such a fragment upstream ofa minimal promoter. A minimal or basal promoter is a piece of DNA whichis capable of recruiting and binding the basal transcription machinery.One example of basal transcription machinery in eukaryotic cells is theRNA polymerase II complex and its accessory proteins. The enzymaticcomponents of the basal transcription machinery are capable ofinitiating and elongating transcription of a given gene, utilizing aminimal or basal promoter. That is, there are not added cis-actingsequences in the promoter region that are capable of recruiting andbinding transcription factors that modulate transcription, e.g.,enhance, repress, render transcription hormone-dependent, etc.Substitutions, deletions, insertions or any combination thereof can becombined to produce a final construct.

[0090] The promoter sequences of the present invention may be modified,for example for expression in other plant systems. In another approach,novel hybrid promoters can be designed or engineered by a number ofmethods. Many promoters contain upstream sequences which activate,enhance or define the strength and/or specificity of the promoter(Atchison, Ann. Rev. Cell Biol. 4:127, 1988). T-DNA genes, for examplecontain “TATA” boxes defining the site of transcription initiation andother upstream elements located upstream of the transcription initiationsite modulate transcription levels (Gelvin, In: Transgenic Plants (Kung,S.-D. and Us,R., eds, San Diego: Academic Press, pp.49-87, 1988).Another chimeric promoter combined a trimer of the octopine synthase(ocs) activator to the mannopine synthase (mas) activator plus promoterand reported an increase in expression of a reporter gene (Min Ni etal., The Plant Journal 7:661, 1995). The upstream regulatory sequencesof the present invention can be used for the construction of suchchimeric or hybrid promoters. Methods for construction of variantpromoters of the present invention include but are not limited tocombining control elements of different promoters or duplicatingportions or regions of a promoter (see for example U.S. Pat. No.5,110,732 and U.S. Pat. No. 5,097,025). Those of skill in the art arefamiliar with the specific conditions and procedures for theconstruction, manipulation and isolation of macromolecules (e.g., DNAmolecules, plasmids, etc.), generation of recombinant organisms and thescreening and isolation of genes, (see for example Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989;Mailga et al., Methods in Plant Molecular Biology, Cold Spring HarborPress, 1995; Birren et al., Genome Analysis: volume 1, Analyzing DNA,(1997), volume 2, Detecting Genes, (1998), volume 3, Cloning Systems,(1999) volume 4, Mapping Genomes, (1999), Cold Spring Harbor, N.Y.).

[0091] The design, construction, and use of chimeric or hybrid promoterscomprising one or more of cis elements of SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, and SEQ ID NO:26, for modulating or regulating the expression ofoperably linked nucleic acid sequences are also encompassed by thepresent invention.

[0092] The promoter sequences, fragments, regions or cis elementsthereof of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNOS:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 are capable oftranscribing operably linked DNA sequences in multiple tissues andtherefore can selectively regulate expression of genes in multipletissues.

[0093] For a number of agronomic traits, transcription of a gene orgenes of interest is desirable in multiple tissues to confer the desiredcharacteristic(s). The availability of suitable promoters that regulatetranscription of operably linked genes in selected target tissues ofinterest is desirable, since it may not be desirable to express a genein every tissue, but only in certain tissues. For example, if onedesires to selectively express a target gene for expression of gene forherbicide tolerance, one may desire expression of the herbicidetolerance gene in vegetative and reproductive tissues. The promotersequences of the present invention are useful for regulating geneexpression in multiple tissues including, but not limited to rapidlygrowing meristematic tissues, male reproductive tissues (androecium)such as pollen, anthers, and filaments, and female reproductive tissues(gynoecium) such as the stigma, style, and ovaries, leaves, sepals, andpetals. The promoters of the present invention therefore have utilityfor expression of herbicide tolerance genes, for example, wheretolerance is desired in multiple tissues and stages of plantdevelopment. The promoter sequences of the present invention haveutility for regulating transcription of any target gene including butnot limited to genes for control of fertility, yield, insect tolerance,fungal tolerance, herbicide tolerance, or any desirable trait ofinterest. Particularly preferred genes include herbicide tolerance genesor insect tolerance genes.

[0094] In one embodiment, the promoters of the present invention haveparticular utility for regulating expression of an herbicide tolerancegene where expression of a gene is desired in multiple tissues. Forexample, the herbicide tolerance gene may confer tolerance to theherbicide glyphosate. Examples of suitable glyphosate tolerance genesinclude, but are not limited to glyphosate resistant EPSP synthase genesor gene products that degrade glyphosate such as, a glyphosateoxidoreductase and phosphonate N-acetyl transferase. It is important tohave a wide variety of choices of 5′ regulatory elements for any plantbiotechnology strategy in order to have suitable regulatory elementsthat are most efficient for the expression profile desired.

[0095] In another embodiment, the promoters of the present inventionhave utility for determining gene function. The function of many genesis unknown and the promoters of the present invention can be used asgenetic elements in a construct to allow a phenotypic evaluation of oneor more genes expressed in a sense or antisense orientation. Thepromoters of the present invention can be components in a plantexpression construct developed for a high throughput assay where highlevels of gene expression in constitutive and reproductive tissues isdesired.

[0096] Any plant can be selected for the identification of genes andregulatory sequences. Examples of suitable plant targets for theisolation of genes and regulatory sequences would include but are notlimited to alfalfa, apple, apricot, Arabidopsis, artichoke, arugula,asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry,broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot,cassava, castorbean, cauliflower, celery, cherry, chicory, cilantro,citrus, clementines, clover, coconut, coffee, corn, cotton, cranberry,cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel,figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit,lettuce, leeks, lemon, lime, Loblolly pine, linseed, mango, melon,mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive,onion, orange, an ornamental plant, palm, papaya, parsley, parsnip, pea,peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum,pomegranate, poplar, potato, pumpkin, quince, radiata pine, radiscchio,radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean,spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweetpotato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf,turnip, a vine, watermelon, wheat, yams, and zucchini. Particularlypreferred plants for the identification of regulatory sequences areArabidopsis, corn, wheat, soybean, and cotton.

[0097] The promoter sequences of the present invention were isolatedfrom Arabidopsis thaliana plant DNA. In a preferred embodiment, aconstruct includes the promoter sequences of the present inventionoperably linked to a transcribable sequence along with suitableterminator and regulatory elements. Such a construct may be transformedinto a suitable target plant of interest. Any plant can be used as asuitable host for nucleic acid constructs comprising the promotersequences of the present invention. Examples of suitable target plantsof interest would include, but are not limited to alfalfa, broccoli,cabbage, canola, cauliflower, corn, cotton, cranberry, cucumber,lettuce, pea, poplar, pine, potato, onion, rice, raspberry, soybean,sugarcane, sugarbeet, sunflower, tomato, and wheat.

Promoter Isolation and Modification Methods

[0098] Any number of methods can be used to isolate fragments of thepromoter sequences disclosed herein. A PCR-based approach can be used toamplify flanking regions from a genomic library of a plant usingpublicly available sequence information. A number of methods are knownto those of skill in the art to amplify unknown DNA sequences adjacentto a core region of known sequence. Methods include but are not limitedto inverse PCR (IPCR), vectorette PCR, Y-shaped PCR and genome walkingapproaches. For the present invention, the nucleic acid molecules wereisolated from Arabidopsis by designing PCR primers based on availablesequence information.

[0099] Nucleic acid fragments can also be obtained by other techniquessuch as by directly synthesizing the fragment by chemical means, as iscommonly practiced by using an automated oligonucleotide synthesizer.Fragments can also be obtained by application of nucleic acidreproduction technology, such as the PCR (polymerase chain reaction)technology by recombinant DNA techniques generally known to those ofskill in the art of molecular biology. Regarding the amplification of atarget nucleic- acid sequence (e.g., by PCR) using a particularamplification primer pair, “stringent PCR conditions” refer toconditions that permit the primer pair to hybridize only to the targetnucleic-acid sequence to which a primer having the correspondingwild-type sequence (or its complement) would bind and preferably toproduce a unique amplification product.

[0100] Those of skill in the art are aware of methods for thepreparation of plant genomic DNA. In one approach, genomic DNA librariescan be prepared from a chosen species by partial digestion with arestriction enzyme and size selecting the DNA fragments within aparticular size range. The genomic DNA can be cloned into a suitableconstruct including but not limited to a bacteriophage, and preparedusing a suitable construct such as a bacteriophage using a suitablecloning kit from any number of vendors (see for example Stratagene, LaJolla Calif. or Gibco BRL, Gaithersburg, Md.).

[0101] In another embodiment, the nucleotide sequences of the promotersdisclosed herein can be modified. Those skilled in the art can createDNA molecules that have variations in the nucleotide sequence. Thenucleotide sequences of the present invention as shown in SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:22, SEQ ID NO:23, SEQID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NOS:27, SEQ ID NO:28, SEQID NO:29, and SEQ ID NO:30 may be modified or altered to enhance theircontrol characteristics. For example, the sequences may be modified byinsertion, deletion or replacement of template sequences in a PCR-basedDNA modification approach. “Variant” DNA molecules are DNA moleculescontaining changes in which one or more nucleotides of a native sequenceis deleted, added, and/or substituted, preferably while substantiallymaintaining promoter function. In the case of a promoter fragment,“variant” DNA can include changes affecting the transcription of aminimal promoter to which it is operably linked. Variant DNA moleculescan be produced, for example, by standard DNA mutagenesis techniques orby chemically synthesizing the variant DNA molecule or a portionthereof.

[0102] In addition to their use in modulating gene expression, thepromoter sequences of the present invention also have utility as probesor primers in nucleic acid hybridization experiments. The nucleic-acidprobes and primers of the present invention can hybridize understringent conditions to a target DNA sequence. The term “stringenthybridization conditions” is defined as conditions under which a probeor primer hybridizes specifically with a target sequence(s) and not withnon-target sequences, as can be determined empirically. The term“stringent conditions” is functionally defined with regard to thehybridization of a nucleic-acid probe to a target nucleic acid (i.e., toa particular nucleic-acid sequence of interest) by the specifichybridization procedure (see for example Sambrook et al., 1989, at9.52-9.55, Sambrook et al., 1989 at 9.47-9.52, 9.56-9.58; Kanehisa,Nucl. Acids Res. 12:203-213, 1984; and Wetmur and Davidson, J. Mol.Biol. 31:349-370, 1968). Appropriate stringency conditions which promoteDNA hybridization are, for example, 6.0×sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in laboratory manualsincluding but not limited to Current Protocols in Molecular Biology,John Wiley & Sons, N.Y., 1989, 6.3.1-6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or either the temperature or the salt concentration may be heldconstant while the other variable is changed. For example, hybridizationusing DNA or RNA probes or primers can be performed at 65° C. in 6×SSC,0.5% SDS, 5×Denhardt's, 100 μg/mL nonspecific DNA (e.g., sonicatedsalmon sperm DNA) with washing at 0.5×SSC, 0.5% SDS at 65° C., for highstringency.

[0103] A nucleic acid molecule is said to be the “complement” of anothernucleic acid molecule if they exhibit complete complementarity. As usedherein, molecules are said to exhibit “complete complementarity” whenevery nucleotide of one of the molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low stringency” conditions. Similarly, the moleculesare said to be “complementary” is they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high stringency” conditions. It is contemplated thatlower stringency hybridization conditions such as lower hybridizationand/or washing temperatures can be used to identify related sequenceshaving a lower degree of sequence similarity if specificity of bindingof the probe or primer to target sequence(s) is preserved. Accordingly,the nucleotide sequences of the present invention can be used for theirability to selectively form duplex molecules with complementarystretches of DNA fragments. Detection of DNA segments via hybridizationis well-known to those of skill in the art, and thus depending on theapplication envisioned, one will desire to employ varying hybridizationconditions to achieve varying degrees of selectivity of probe towardstarget sequence and the method of choice will depend on the desiredresults. Conventional stringency conditions are described in Sambrook,et al., Molecular Cloning, A Laboratory Manual, 2^(nd) Ed., Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1989, and by Haymes et al.,Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C., 1985.

[0104] In one embodiment of the present invention, the nucleic acidsequences SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, or afragment, region, cis element, or oligomer of these sequences, are usedin hybridization assays of other plant tissues to identify closelyrelated or homologous genes and associated regulatory sequences. Theseinclude but are not limited to Southern or northern hybridization assayson any substrate including but not limited to an appropriately preparedplant tissue, cellulose, nylon, or combination filter, chip, or glassslide. Such methodologies are well known in the art and are available ina kit or preparation which can be supplied by commercial vendors.

[0105] A fragment of a nucleic acid as used herein is a portion of thenucleic acid that is less than full-length. For example, for the presentinvention any length of nucleotide sequence that is less than thedisclosed nucleotide sequences of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, and SEQ ID NO:26 is considered to be a fragment. A fragment canalso comprise at least a minimum length capable of hybridizingspecifically with a native nucleic acid under stringent hybridizationconditions as defined above. The length of such a minimal fragment ispreferably at least 8 nucleotides, more preferably 15 nucleotides, evenmore preferably at least 20 nucleotides, and most preferably at least 30nucleotides of a native nucleic acid sequence.

[0106] A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, or enzyme. “Primers” areisolated nucleic acids that are annealed to a complementary target DNAstrand by nucleic acid hybridization to form a hybrid between the primerand the target DNA strand, then extended along the target DNA strand bya polymerase, e.g., a DNA polymerase. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other conventional nucleic-acid amplification methods.

[0107] Probes and primers are generally 11 nucleotides or more inlength, preferably 18 nucleotides or more, more preferably 25nucleotides, and most preferably 30 nucleotides or more. Such probes andprimers hybridize specifically to a target DNA or RNA sequence underhigh stringency hybridization conditions and hybridize specifically to atarget native sequence of another species under lower stringencyconditions. Preferably, probes and primers according to the presentinvention have complete sequence similarity with the native sequence,although probes differing from the native sequence and that retain theability to hybridize to target native sequences may be designed byconventional methods. Methods for preparing and using probes and primersare described, for example, in Molecular Cloning: A Laboratory Manual,2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989 (hereinafter, “Sambrook et al.,1989”); Current Protocols in Molecular Biology, ed. Ausubel et al.,Greene Publishing and Wiley-Interscience, N.Y., 1992 (with periodicupdates) (hereinafter, “Ausubel et al., 1992); and Innis et al., PCRProtocols: A Guide to Methods and Applications, Academic Press: SanDiego, 1990. PCR-primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5, © 1991, Whitehead Institute for BiomedicalResearch, Cambridge, Mass.). Primers and probes based on the nativepromoter sequences disclosed herein can be used to confirm and, ifnecessary, to modify the disclosed sequences by conventional methods,e.g., by re-cloning and re-sequencing.

Constructs and Expression Constructs

[0108] Native or synthetic nucleic acids according to the presentinvention can be incorporated into recombinant nucleic acid constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. In a preferred embodiment, the nucleotide sequences ofthe present invention as shown in SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NOS:27, SEQ ID NO:28, SEQ ID NO:29, and SEQID NO:30 or fragments, variants or derivatives thereof are incorporatedinto an expression cassette which includes the promoter regions of thepresent invention operably linked to a genetic component such as aselectable, screenable, or scorable marker gene.

[0109] In another embodiment, the disclosed nucleic acid sequences ofthe present invention as shown in SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NOS:27, SEQ ID NO:28, SEQ ID NO:29, and SEQID NO:30 are operably linked to a genetic component such as a nucleicacid which confers a desirable characteristic associated with plantmorphology, physiology, growth and development, yield, nutritionalenhancement, disease or pest resistance, or environmental or chemicaltolerance. These genetic components such as marker genes or agronomicgenes of interest can function in the identification of a transformedplant cell or plant, or a produce a product of agronomic utility.

[0110] In another embodiment, one genetic component produces a productwhich serves as a selection device and functions in a regenerable planttissue to produce a compound which would confer upon the plant tissueresistance to an otherwise toxic compound. Genes of interest for use asa selectable, screenable, or scorable marker would include but are notlimited to GUS (coding sequence for beta-glucuronidase), GFP (codingsequence for green fluorescent protein), LUX (coding gene forluciferase), antibiotic resistance marker genes, or herbicide tolerancegenes. Examples of transposons and associated antibiotic resistancegenes include the transposons Tns (bla), Tn5 (nptII), Tn7 (dhfr),penicillins, kanamycin (and neomycin, G418, bleomycin); methotrexate(and trimethoprim); chloramphenicol; kanamycin and tetracycline.

[0111] Characteristics useful for selectable markers in plants have beenoutlined in a report on the use of microorganisms (Advisory Committee onNovel Foods and Processes, July 1994). These include stringent selectionwith minimum number of nontransformed tissues, large numbers ofindependent transformation events with no significant interference withthe regeneration, application to a large number of species, andavailability of an assay to score the tissues for presence of themarker.

[0112] A number of selectable marker genes are known in the art andseveral antibiotic resistance markers satisfy these criteria, includingthose resistant to kanarnycin (nptII), hygromycin B (aph IV) andgentamycin (aac3 and aacC4). Useful dominant selectable marker genesinclude genes encoding antibiotic resistance genes (e.g., resistance tohygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin);and herbicide resistance genes (e.g., phosphinothricinacetyltransferase). A useful strategy for selection of transformants forherbicide resistance is described, e.g., in Vasil, Cell Culture andSomatic Cell Genetics of Plants, Vols. I-III, Laboratory Procedures andTheir Applications Academic Press, N.Y., 1984. Particularly preferredselectable marker genes for use in the present invention would geneswhich confer resistance to compounds such as antibiotics like kanamycin,and herbicides like glyphosate (Della-Cioppa et al., Bio/Technology5(6), 1987, U.S. Pat. No. 5,463,175, U.S. Pat. No. 5,633,435). Otherselection devices can also be implemented and would still fall withinthe scope of the present invention.

[0113] For the practice of the present invention, conventionalcompositions and methods for preparing and using DNA constructs and hostcells are employed, as discussed, inter alia, in Sambrook et al., 1989.In a preferred embodiment, the host cell is a plant cell. A number ofDNA constructs suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987);Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990; and R.R.D. Croy Plant Molecular BiologyLabFax, BIOS Scientific Publishers, 1993. Plant expression constructscan include, for example, one or more cloned plant genes under thetranscriptional control of 5′ and 3′ regulatory sequences. They can alsoinclude a selectable marker as described to select for host cellscontaining the expression construct. Such plant expression constructsalso contain a promoter regulatory region (e.g., a regulatory regioncontrolling inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and apolyadenylation signal. Other sequences of bacterial origin are alsoincluded to allow the construct to be cloned in a bacterial host. Theconstruct will also typically contain a broad host range prokaryoticorigin of replication. In a particularly preferred embodiment, the hostcell is a plant cell and the plant expression construct comprises apromoter region as disclosed in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NOS:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ IDNO:30; an operably linked transcribable sequence; and a transcriptiontermination sequence. Other regulatory sequences envisioned as geneticcomponents in an expression construct include but is not limited tonon-translated leader sequence which can be coupled with the promoter.In a particularly preferred embodiment, the host cell is a plant celland the plant expression construct comprises a promoter region asdisclosed in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNOS:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; an operably linkedtranscribable sequence, and a transcription termination sequence. Plantexpression constructs also can comprise additional sequences includingbut not limited to polylinker sequences that contain restriction enzymesites that are useful for cloning purposes.

Genetic Elements in Plant Expression Constructs

[0114] Plant expression constructs may include more than one expressiblegene sequence, each operably linked to a different promoter. A number ofpromoters have utility for plant gene expression for any gene ofinterest including but not limited to selectable markers, scorablemarkers, genes for pest tolerance, disease tolerance, nutritionalenhancements and any other gene of agronomic interest. Examples ofconstitutive promoters useful for plant gene expression include but arenot limited to, the cauliflower mosaic virus (CaMV) P-35S promoter,which confers constitutive, high-level expression in most plant tissues(see, e.g., Odel et al., Nature 313:810, 1985), including monocots (see,e.g., Dekeyser et al., Plant Cell 2:591, 1990; Terada and Shimamoto,Mol. Gen. Genet. 220:389, 1990); a tandemly duplicated version of theCaMV 35S promoter, the enhanced 35S promoter (P-e35S) the nopalinesynthase promoter (An et al., Plant Physiol. 88:547, 1988), the octopinesynthase promoter (Fromm et al., Plant Cell 1:977, 1989); and thefigwort mosaic virus (P-FMV) promoter as described in U.S. Pat. No.5,378,619 and an enhanced version of the FMV promoter (P-eFMV) where thepromoter sequence of P-FMV is duplicated in tandem, the cauliflowermosaic virus 19S promoter, a sugarcane bacilliform virus promoter, acommelina yellow mottle virus promoter, and other plant DNA viruspromoters known to express in plant cells.

[0115] A variety of plant gene promoters that are regulated in responseto environmental, hormonal, chemical, and/or developmental signals canbe used for expression of an operably linked gene in plant cells,including promoters regulated by (1) heat (Callis et al., Plant Physiol.88:965, 1988), (2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al.,Plant Cell 1:471, 1989; maize rbcS promoter, Schaffner and Sheen, PlantCell 3:997, 1991; or chlorophyll a/b-binding protein promoter, Simpsonet al., EMBO J. 4:2723, 1985), (3) hormones, such as abscisic acid(Marcotte et al., Plant Cell 1:969, 1989), (4) wounding (e.g., wuni,Siebertz et al., Plant Cell 1:961, 1989); or (5) chemicals such asmethyl jasmonate, salicylic acid, or Safener. It may also beadvantageous to employ (6) organ-specific promoters (e.g., Roshal etal., EMBO J. 6:1155, 1987; Schernthaner et al., EMBO J. 7:1249, 1988;Bustos et al., Plant Cell 1:839, 1989). The promoters of the presentinvention are plant promoters that are capable of transcribing operablylinked DNA sequences in rapidly growing meristematic tissue andreproductive tissues and can be operably linked to any gene of interestin an expression construct.

[0116] Plant expression constructs can include RNA processing signals,e.g., introns, which may be positioned upstream or downstream of apolypeptide-encoding sequence in the transgene. In addition, theexpression constructs may include additional regulatory sequences fromthe 3′-untranslated region of plant genes (Thornburg et al., Proc. Natl.Acad. Sci. USA 84:744 (1987); An et al., Plant Cell 1:115 (1989), e.g.,a 3′ terminator region to increase mRNA stability of the mRNA, such asthe PI-II terminator region of potato or the octopine or nopalinesynthase 3′ terminator regions. 5′ non-translated regions of a MRNA canplay an important role in translation initiation and can also be agenetic component in a plant expression construct. For example,non-translated 5′ leader sequences derived from heat shock protein geneshave been demonstrated to enhance gene expression in plants (see, forexample U.S. Pat. No. 5,362,865). These additional upstream anddownstream regulatory sequences may be derived from a source that isnative or heterologous with respect to the other elements present on theexpression construct.

[0117] The promoter sequences of the present invention are used tocontrol gene expression in plant cells. The disclosed promoter sequencesare genetic components that are part of constructs used in planttransformation. The promoter sequences of the present invention can beused with any suitable plant transformation plasmid or constructcontaining a selectable or screenable marker and associated regulatoryelements, as described, along with one or more nucleic acids expressedin a manner sufficient to confer a particular desirable trait. Examplesof suitable structural genes of agronomic interest envisioned by thepresent invention would include but are not limited to one or more genesfor insect tolerance, such as a Bacillus thuringiensis (B.t.) gene, pesttolerance such as genes for fungal disease control, herbicide tolerancesuch as genes conferring glyphosate tolerance, and genes for qualityimprovements such as yield, nutritional enhancements, environmental orstress tolerances, or any desirable changes in plant physiology, growth,development, morphology or plant product(s). For example, structuralgenes would include any gene that confers insect tolerance including butnot limited to a Bacillus insect control protein gene as described in WO9931248, herein incorporated by reference in its entirety, U.S. Pat. No.5,689,052, herein incorporated by reference in its entirety, U.S. Pat.Nos. 5,500,365 and 5,880275, herein incorporated by reference it theirentirety. In another embodiment, the structural gene can confertolerance to the herbicide glyphosate as conferred by genes including,but not limited to Agrobacterium strain CP4 glyphosate resistant EPSPSgene (aroA:CP4) as described in U.S. Pat. No. 5,633,435, hereinincorporated by reference in its entirety, or glyphosate oxidoreductasegene (GOX) as described in U.S. Pat. No. 5,463,175, herein incorporatedby reference in its entirety.

[0118] Alternatively, the DNA coding sequences can effect thesephenotypes by encoding a non-translatable RNA molecule that causes thetargeted inhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev. 9:207,1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous MRNA product (see for example, Gibson and Shillitoe, Mol.Biotech. 7:125,1997). Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention.

[0119] In addition to regulatory elements or sequences located upstream(5′) or within a DNA sequence, there are downstream (3′) sequences thataffect gene expression. Thus, the term regulatory sequence as usedherein refers to any nucleotide sequence located upstream, within, ordownstream to a DNA sequence which controls, mediates, or affectsexpression of a gene product in conjunction with the protein syntheticapparatus of the cell.

[0120] Those of skill in the art are aware of the constructs suitablefor plant transformation. The promoter sequences of the presentinvention are preferably incorporated into an expression construct usingscreenable or scorable markers as described and tested in transientanalyses to provide an indication of gene expression in transformedplants. Methods of testing gene expression in transient assays are knownto those of skill in the art. Transient expression of marker genes hasbeen reported using a variety of plants, tissues and DNA deliverysystems. For example, types of transient analyses can include but arenot limited to direct gene delivery via electroporation or particlebombardment of tissues in any transient plant assay using any plantspecies of interest. Such transient systems would include but are notlimited to protoplasts from suspension cultures in wheat (Zhou et al.,Plant Cell Reports 12:612. 1993), electroporation of leaf protoplasts ofwheat (Sethi et al., J. Crop Sci. 52: 152, 1983); electroporation ofprotoplast prepared from corn tissue (Sheen, J. The Plant Cell 3: 225,1991), or particle bombardment of specific tissues of interest. Thepresent invention encompasses the use of any transient expression systemto evaluate regulatory sequences operatively linked to selected reportergenes, marker genes or agronomic genes of interest. Examples of planttissues envisioned to test in transients via an appropriate deliverysystem would include but are not limited to leaf base tissues, callus,cotyledons, roots, endosperm, embryos, floral tissue, pollen, andepidermal tissue.

[0121] Any scorable or screenable marker can be used in a transientassay. Preferred marker genes for transient analyses of the promoters or5′ regulatory sequences of the present invention include aP-glucuronidase (GUS) gene or a green fluorescent protein (GFP) gene.The expression constructs containing the 5′ regulatory sequencesoperably linked to a marker gene are delivered to the tissues and thetissues are analyzed by the appropriate mechanism, depending on themarker. The quantitative or qualitative analyses are used as a tool toevaluate the potential expression profile of the 5′ regulatory sequenceswhen operatively linked to genes of agronomic interest in stable plants.Ultimately, the 5′ regulatory sequences of the present invention aredirectly incorporated into suitable plant transformation expressionconstructs comprising the 5′ regulatory sequences operatively linked toa transcribable DNA sequence interest, transformed into plants and thestably transformed plants and progeny thereof analyzed for the desiredexpression profile conferred by the 5′ regulatory sequences.

[0122] Suitable expression constructs for introducing exogenous DNA intoplant cells would include but are not limited to disarmed Ti-plasmidsfor Agrobacterium-mediated methods. These constructs can contain aresistance marker, 1-2 T-DNA borders, and origins of replication for E.coli and Agrobacterium along with one or more genes of interest andassociated regulatory regions. Those of skill in the art are aware thatfor Agrobacterium-mediated approaches a number of strains and methodsare available. Such strains would include but are not limited toAgrobacterium strains C58, LBA4404, EHA101 and EHA105. Particularlypreferred strains are Agrobacterium tumefaciens strains.

[0123] Exemplary nucleic acids which may be introduced by the methodsencompassed by the present invention include, for example, DNA sequencesor genes from another species, or even genes or sequences whichoriginate with or are present in the same species, but are incorporatedinto recipient cells by genetic engineering methods rather thanclassical reproduction or breeding techniques. However, the term“exogenous” is also intended to refer to genes that are not normallypresent in the cell being transformed, or perhaps simply not present inthe form, structure, etc., as found in the transforming DNA segment orgene, or genes which are normally present and that one desires toexpress in a manner that differs from the natural expression pattern,e.g., to over-express. Thus, the term “exogenous” gene or DNA isintended to refer to any gene or DNA segment that is introduced into arecipient cell, regardless of whether a similar gene may already bepresent in such a cell. The type of DNA included in the exogenous DNAcan include DNA which is already present in the plant cell, DNA fromanother plant, DNA from a different organism, or a DNA generatedexternally, such as a DNA sequence containing an antisense message of agene, or a DNA sequence encoding a synthetic or modified version of agene.

[0124] The plant transformation constructs containing the promotersequences of the present invention may be introduced into plants by anyplant transformation method. Several methods are available forintroducing DNA sequences into plant cells and are well known in theart. Suitable methods include but are not limited to bacterial infection(e.g., with Agrobacterium as described above), binary bacterialartificial chromosome constructs, direct delivery of DNA (e.g. viaPEG-mediated transformation, desiccation/inhibition-mediated DNA uptake,electroporation, agitation with silicon carbide fibers), andacceleration of DNA coated particles (reviewed in Potrykus, Ann. Rev.Plant Physiol. Plant Mol. Biol., 42: 205, 1991).

[0125] Methods for specifically transforming dicots primarily useAgrobacterium tumefaciens. For example, transgenic plants reportedinclude but are not limited to cotton (U.S. Pat. No. 5,004,863; U.S.Pat. No. 5,159,135; U.S. Pat. No. 5,518,908, WO 97/43430), soybean (U.S.Pat. No. 5,569,834; U.S. Pat. No. 5,416,011; McCabe et al.,Bio/Technology, 6:923, 1988; Christou et al., Plant Physiol., 87:671,1988); Brassica (U.S. Pat. No. 5,463,174), and peanut (Cheng et al.,Plant Cell Rep., 15: 653, 1996).

[0126] Similar methods have been reported in the transformation ofmonocots. Transformation and plant regeneration using these methods havebeen described for a number of crops including but not limited toasparagus (Asparagus officinalis; Bytebier et al., Proc. Natl. Acad.Sci. U.S.A., 84: 5345, 1987); barley (Hordeum vulgarae; Wan and Lemaux,Plant Physiol., 104: 37, 1994); maize (Zea mays; Rhodes, C. A., et al.,Science, 240: 204, 1988; Gordon-Kamm, et al., Plant Cell, 2: 603, 1990;Fromm, et al., Bio/Technology, 8: 833, 1990; Koziel, et al.,Bio/Technology, 11: 194, 1993); oats (Avena sativa; Somers, et al.,Bio/Technology, 10: 1589, 1992); orchardgrass (Dactylis glomerata; Horn,et al., Plant Cell Rep., 7: 469, 1988); rice (Oryza sativa, includingindica and japonica varieties, Toriyama, et al., Bio/Technology, 6: 10,1988; Zhang, et al., Plant Cell Rep., 7: 379, 1988; Luo and Wu, PlantMol. Biol. Rep., 6: 165, 1988; Zhang and Wu, Theor. Appl. Genet., 76:835, 1988; Christou, et al., Bio/Technology, 9: 957, 1991); sorghum(Sorghum bicolor; Casas, A.M., et al., Proc. Natl. Acad. Sci. U.S.A.,90: 11212, 1993); sugar cane (Saccharum spp.; Bower and Birch, Plant J.,2: 409, 1992); tall fescue (Festuca arundinacea; Wang, Z. Y. et al.,Bio/Technology, 10: 691, 1992); turfgrass (Agrostis palustris; Zhong etal., Plant Cell Rep., 13: 1, 1993); wheat (Triticum aestivum; Vasil etal., Bio/Technology, 10: 667, 1992; Weeks T., et al., Plant Physiol.,102: 1077, 1993; Becker, et al., Plant, J. 5: 299, 1994), and alfalfa(Masoud, S. A., et al., Transgen. Res., 5: 313, 1996). It is apparent tothose of skill in the art that a number of transformation methodologiescan be used and modified for production of stable transgenic plants fromany number of target crops of interest.

Plant Analysis Methods

[0127] The transformed plants are analyzed for the presence of the genesof interest and the expression level and/or profile conferred by thepromoter sequences of the present invention. Those of skill in the artare aware of the numerous methods available for the analysis oftransformed plants. A variety of methods are used to assess geneexpression and determine if the introduced gene(s) is integrated,functioning properly, and inherited as expected. For the presentinvention the promoters can be evaluated by determining the expressionlevels of genes to which the promoters are operatively linked. Apreliminary assessment of promoter function can be determined by atransient assay method using reporter genes, but a more definitivepromoter assessment can be determined from the analysis of stableplants. Methods for plant analysis include but are not limited toSouthern blots or northern blots, PCR-based approaches, biochemicalanalyses, phenotypic screening methods, field evaluations, andimmunodiagnostic assays.

[0128] The methods of the present invention including but not limited toPCR technologies, genomic DNA isolation, expression constructconstruction, transient assays, and plant transformation methods arewell known to those of skill in the art and are carried out usingstandard techniques or modifications thereof.

Glyphosate Spray Tests

[0129] In one embodiment a greenhouse or field evaluation for glyphosatetolerance is conducted. The term “glyphosate” is used herein to refercollectively to the parent herbicide N-phosphonomethylglycine (otherwiseknown as glyphosate acid), to a salt or ester thereof, or to a compoundwhich is converted to N-phosphonomethylglycine in plant tissues or whichotherwise provides N-phosphonomethylglycine in ionic form (otherwiseknown as glyphosate ion). Illustratively, water-soluble glyphosate saltsuseful herein are disclosed in U.S. Pat. Nos. 3,799,758 and 4,405,531 toFranz, the disclosure of which is incorporated herein by reference.Glyphosate salts that can be used according to the present inventioninclude but are not restricted to alkali metal, for example sodium andpotassium, salts; ammonium salt; C₁₋₁₆ alkylammonium, for exampledimethylammonium and isopropylamrnonium, salts; C₁₋₁₆ alkanolammonium,for example monoethanolammonium, salt; C₁₋₁₆ alkylsulfonium, for exampletrimethylsulfonium, salts; mixtures thereof and the like. The glyphosateacid molecule has three acid sites having different pKa values;accordingly mono-, di- and tribasic salts, or any mixture thereof, orsalts of any intermediate level of neutralization, can be used.

[0130] Glyphosate salts are commercially significant in part becausethey are water-soluble. Many ammonium, alkylammonium, alkanolammonium,alkylsulfonium and alkali metal salts are highly water-soluble, allowingfor formulation as highly concentrated aqueous solutions which can bediluted in water at the point of use.

[0131] Such concentrated aqueous solutions can contain about 50 to about500 grams per liter of glyphosate, expressed as acid equivalent (ga.e./1). Higher glyphosate concentrations, for example about 300 toabout 500 g a.e./1, are preferred.

[0132] Glyphosate salts are alternatively formulated as water-soluble orwater-dispersible compositions, in the form for example of powders,granules, pellets or tablets. Such compositions are often known as dryformulations, although the term “dry” should not be understood in thiscontext to imply the complete absence of water. Typically, dryformulations contain less than about 5% by weight of water, for exampleabout 0.5% to about 2% by weight of water. Such formulations areintended for dissolution or dispersion in water at the point of use.

[0133] Contemplated dry glyphosate formulations can contain about 5% toabout 80% by weight of glyphosate, expressed as acid equivalent (%a.e.). Higher glyphosate concentrations within the above range, forexample about 50% to about 80% a.e., are preferred. Especially usefulsalts of glyphosate for making dry formulations are sodium and ammoniumsalts.

[0134] Plant treatment compositions and liquid and dry concentratecompositions of the invention can optionally contain one or more desiredexcipient ingredients. Especially useful excipient ingredients forglyphosate compositions are surfactants, which assist in retention ofaqueous spray solutions on the relatively hydrophobic surfaces of plantleaves, as well as helping the glyphosate to penetrate the waxy outerlayer (cuticle) of the leaf and thereby contact living tissues withinthe leaf. Surfactants can perform other useful functions as well.

[0135] There is no restriction in the type or chemical class ofsurfactant that can be used in glyphosate compositions of the invention.Nonionic, anionic, cationic and amphoteric types, or combinations ofmore than one of these types, are all useful in particular situations.However, it is generally preferred that at least one of the surfactants,if any, present should be other than anionic; i.e., at least one of thesurfactants should be nonionic, cationic or amphoteric.

[0136] Many surfactants useful herein have a chemical structure thatcomprises one or more moieties each consisting of a single C₂₋₄ alkyleneoxide unit or a polymerized or copolymerized chain of C₂₋₄ alkyleneoxide units. Such surfactants are referred to as polyoxyalkylenesurfactants and include nonionic, anionic, cationic and amphoterictypes. Polyoxyalkylene surfactants useful in presently contemplatedcompositions contain about 2 to about 100 C₂₋₄ alkylene oxide units. Inpreferred polyoxyalkylene surfactants the alkylene oxide units form oneor more chains of either ethylene oxide or copolymerized ethylene oxideand propylene oxide, each chain of alkylene oxide units having aterminal hydrido group or a C₁₋₄ alkyl or C₁₋₄ alkanoyl end-cap.

[0137] Hydrophobic moieties of surfactants useful in compositions of theinvention can be essentially hydrocarbon based, in which case thehydrophobic moieties are typically C₈₋₂₄, preferably C₁₂₋₁₈, alkyl,alkenyl, alkylaryl, alkanoyl or alkenoyl chains. These chains can belinear or branched. Alternatively, the hydrophobic moieties can containsilicon atoms, for example in the form of siloxane groups such asheptamethyltrisiloxane groups, or fluorine atoms, for example aspartially-fluorinated alkyl or perfluoroalkyl chains.

[0138] Among nonionic surfactants, especially preferred classes includepolyoxyethylene alkyl, alkenyl or alkylaryl ethers, such as ethoxylatedprimary or secondary alcohols or alkylphenols, polyoxyethylene alkyl oralkenyl esters, such as ethoxylated fatty acids, polyoxyethylenesorbitan alkyl esters, glyceryl alkyl esters, sucrose esters, alkylpolyglycosides, and the like. Representative specific examples of suchnonionic surfactants include polyoxyethylene (9) nonylphenol, Neodol™25-7 of Shell (a polyoxyethylene (7) C₁₂₋₁₅ linear primary alcohol),Tergitol™ 15-S-9 of Union Carbide (a polyoxyethylene (9) C₁₂₋₁₅secondary alcohol), Tween™ 20 of ICI (a polyoxyethylene (20) sorbitanmonolaurate) and Agrimul™ PG-2069 of Henkel (a C₉₋₁₁ alkylpolyglucoside).

[0139] Among cationic surfactants, especially preferred classes includepolyoxyethylene tertiary alkylamines or alkenylamines, such asethoxylated fatty amines, quaternary ammonium surfactants,polyoxyethylene alkyletheramines, and the like. Representative specificexamples of such cationic surfactants include polyoxyethylene (5)cocoamine, polyoxyethylene (15) tallowamine, distearyldimethylammoniumchloride, cetyltrimethylammonium bromide, methylbis(2-hydroxyethyl)cocoammonium chloride, N-dodecylpyridine chloride andpolyoxypropylene (8) ethoxytrimethylammonium chloride. Particularlypreferred polyoxyethylene alkyletheramines are those disclosed in PCTPublication No. WO 96/32839. Many cationic quaternary ammoniumsurfactants of diverse structures are known in the art to be useful incombination with glyphosate and can be used in compositions contemplatedherein; such quaternary ammonium surfactants have the formula

(NR^(a)R^(b)R^(c)R^(d))m^(A)n

[0140] where A is a suitable anion such as chloride, bromide, iodide,acetate, sulfate or phosphate, m and n are integers such that thepositive electrical charges on cations (NR^(a)R^(b)R^(c)R^(d)) balancethe negative electrical charges on anions A, and options for R^(a),R^(b), R^(c) and R^(d) include, without limitation:

[0141] (i) R^(a) is benzyl or C₈₋₂₄, preferably C₁₂₋₁₈, alkyl oralkenyl, and R^(b), R^(c) and R^(d) are independently C₁₋₄ alkyl,preferably methyl;

[0142] (ii) R^(a) and R^(b) are independently C₈₋₂₄, preferably C₁₂₋₁₈,alkyl or alkenyl, and R^(c) and R^(d) are independently C₁₋₄ alkyl,preferably methyl;

[0143] (iii) R^(a) is C₈₋₂₄, preferably C₁₂₋₁₈, alkyl or alkenyl, R^(b)is a polyoxyalkylene chain having about 2 to about 100 C₂₋₄ alkyleneoxide units, preferably ethylene oxide units, and R^(c) and R^(d) areindependently C₁₋₄ alkyl, preferably methyl;

[0144] (iv) R^(a) is C₈₋₂₄, preferably C₁₂₋₁₈, alkyl or alkenyl, R^(b)and R^(c) are polyoxyalkylene chains having in total about 2 to about100 C₂₋₄ alkylene oxide units, preferably ethylene oxide units, andR^(d) is C₁₋₄ alkyl, preferably methyl; or

[0145] (v) R^(a) is a polyoxyalkylene chain having about 2 to about 100C₂₋₄ alkylene oxide units in which C₃₋₄ alkylene oxide units, preferablypropylene oxide units, predominate and R^(b), R^(c) and R^(d) areindependently C₁₋₄ alkyl, preferably methyl or ethyl.

[0146] Particularly preferred quaternary ammonium surfactants of thistype are those disclosed in U.S. Pat. No. 5,464,807 to Claude et al.

[0147] In one embodiment, the anion A associated with such a quaternaryammonium surfactant can be a glyphosate anion.

[0148] Among amphoteric surfactants, including as is customary in theart surfactants more correctly described as zwitterionic, especiallypreferred classes include polyoxyethylene alkylamine oxides,alkylbetaines, alkyl-substituted amino acids and the like Representativeexamples of such amphoteric surfactants include dodecyldimethylamineoxide, polyoxyethylene (2) cocoamine oxide and stearyldimethylbetaine.

[0149] Standard reference sources from which one of skill in the art canselect suitable surfactants, without limitation to the above mentionedclasses, include Handbook of Industrial Surfactants, Second Edition(1997) published by Gower, McCutcheon's Emulsifiers and Detergents,North American and International Editions (1997) published by MCPublishing Company, and International Cosmetic Ingredient Dictionary,Sixth Edition (1995) Volumes 1 and 2, published by the Cosmetic,Toiletry and Fragrance Association.

[0150] Other optional components of compositions of the inventioninclude agents to modify color, viscosity, gelling properties, freezingpoint, hygroscopicity, caking behavior, dissolution rate,dispersibility, or other formulation characteristics.

[0151] Examples of commercial formulations of glyphosate include,without restriction, those sold by Monsanto Company as ROUNDUP®,ROUNDUP® ULTRA, ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP® BIACTIVE, ROUNDUP®BIOFORCE, RODEO®), POLARIS(®, SPARK® and ACCORD® herbicides, all ofwhich contain glyphosate as its isopropylammonium salt; those sold byMonsanto Company as ROUNDUP® DRY and RIVAL® herbicides, which containglyphosate as its ammonium salt; that sold by Monsanto Company asROUNDUP® GEOFORCE, which contains glyphosate as its sodium salt; andthat sold by Zeneca Limited as TOUCHDOWN® herbicide, which containsglyphosate as its trimethylsulfonium salt.

[0152] The selection of application rates for a glyphosate formulationthat are biologically effective is within the skill of the ordinaryagricultural technician. One of skill in the art will likewise recognizethat individual plant conditions, weather conditions and growingconditions can affect the results achieved in practicing the process ofthe present invention. Over two decades of glyphosate use and publishedstudies relating to such use have provided abundant information fromwhich a weed control practitioner can select glyphosate applicationrates that are herbicidally effective on particular species atparticular growth stages in particular environmental conditions.

[0153] A process of the present invention is applicable to any and allplant species on which glyphosate is biologically effective as aherbicide or plant growth regulator. This encompasses a very widevariety of plant species worldwide. Likewise, compositions of theinvention can be applied to any and all plant species on whichglyphosate is biologically effective.

[0154] In one embodiment, a glyphosate-containing herbicide is appliedto the plant comprising the DNA constructs of the present invention, andthe plants are evaluated for tolerance to the glyphosate herbicide. Anyformulation of glyphosate can be used for testing plants comprising theDNA constructs of the present invention. For example, a glyphosatecomposition such as Roundup Ultra™ can be used. The testing parametersfor an evaluation of the glyphosate tolerance of the plant will varydepending on a number of factors. Factors would include, but are notlimited to the type of glyphosate formulation, the concentration andamount of glyphosate used in the formulation, the type of plant, theplant developmental stage during the time of the application,environmental conditions, the application method, and the number oftimes a particular formulation is applied. For example, plants can betested in a greenhouse environment using a spray application method. Thetesting range using Roundup Ultra™ can include, but is not limited to 8oz/acre to 256 oz/acre. The preferred commercially effective range canbe from 16 oz/acre to 64 oz/acre of Roundup Ultra™, depending on thecrop and stage of plant development. A crop can be sprayed with at leastone application of a glyphosate formulation. For testing in cotton anapplication of 32 oz/acre at the 3-leaf stage may be followed byadditional applications at later stages in development. For wheat anapplication of 32 oz/acre of Roundup Ultra™ at the 3-5 leaf stage can beused and may be followed with a pre- or post-harvest application,depending on the type of wheat to be tested. The test parameters can beoptimized for each crop in order to find the particular plant comprisingthe constructs of the present invention that confers the desiredcommercially effective glyphosate tolerance level.

[0155] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention, therefore all matter set forth or shown in theaccompanying drawings is to be interpreted as illustrative and not in alimiting sense.

EXAMPLES Example 1

[0156] The plasmid constructs used are either pUC cloning constructs ordouble border plant transformation constructs containing an E. coliorigin of replication such as ori322, a broad host range origin ofreplication such as oriV or oriRi, and a coding region for a selectablemarker such as Spc/Str that encodes for Tn7 aminoglycosideadenyltransferase (aadA) confers resistance to spectinomycin orstreptomycin, or a gentamicin (Gm, Gent) selectable marker. For planttransformation, the host bacterial strain was Agrobacterium tumefaciensABI or LBA4404.

[0157] The genetic elements are described as follows: P-e35S is the 35SRNA from CaMV containing a duplication of the −90−300 region asdescribed in U.S. Pat. No. 5,424,200 herein incorporated by reference inits entirety; P-FMV is the 34S promoter from Figwort Mosaic Virus asdescribed in U.S. Pat. No. 5,378,619 herein incorporated by reference inits entirety; P-eFMV is a derivative of the FMV promoter containing aduplicated enhancer region of the FMV promoter; CTP2 is the transitpeptide region of Arabidopsis EPSP synthase as described in U.S. Pat.No. 5,633,435; aroA:CP4syn (aroA:CP4) is the coding region for CP4 EPSP(synthetic sequence) as described in U.S. Pat. No. 5,633,435 or furthermodified for expression in plants based on codon usage of particularplant species; E9 3′ is the 3′ end of an isolate of the pea RbcS genethat functions as a polyadenylation signal; nos is the 3′ end of thenopaline synthase gene that functions as a polyadenylation signal; Hsp70is the non-translated leader sequence from Petunia hybrida as describedin U.S. Pat. No. 5,362,865 herein incorporated by reference in itsentirety; GUS is the beta-glucuronidase coding sequence from E. coli(Jefferson, R. A. Proc. Natl. Acad. Sci. U.S.A., 83:8447-8451, 1987);the right border (RB) and left borders (LB) are from the Ti plasmid ofAgrobacterium tumefaciens octopine and nopaline strains. The P-AtAct2 isthe promoter from the Arabidopsis thaliana actin 2 gene; AtAct2i is theintron in the 5′ untranslated region (UTR) of the Arabidopsis thalianaactin 2 gene; P-AtAct8 is the promoter from the Arabidopsis thalianaactin 8 gene; AtAct2i is the intron in the 5′ UTR of the Arabidopsisthaliana actin 8 gene; P-AtAct11 is the promoter from the Arabidopsisthaliana actin 11 gene; AtAct11i is the intron in the 5′ UTR of theArabidopsis thaliana actin 11 gene; P-AtAct1a is the promoter from theArabidopsis thaliana actin 1a gene, L-AtAct1a is the untranslated leaderand I-AtAct1a is the intron from the genomic DNA of the actin 1a gene;P-AtAct1b is the promoter from the Arabidopsis thaliana actin 1b gene,L-AtAct1b is the untranslated leader and I-AtAct1b is the intron fromthe genomic DNA of the actin 1b gene; P-AtAct3 is the promoter from theArabidopsis thaliana actin 3 gene, L-AtAct3 is the untranslated leaderand I-AtAct3 is the intron from the genomic DNA of the actin 3 gene;P-AtAct7 is the promoter from the Arabidopsis thaliana actin 7 gene,L-AtAct7 is the untranslated leader and I-AtAct7 is the intron from thegenomic DNA of the actin 7 gene; P-AtAct12 is the promoter from theArabidopsis thaliana actin 12 gene, L-AtAct12 is the untranslated leaderand I-AtAct12 is the intron from the genomic DNA of the actin 12 gene;P-AtEF1α (P-AtEF1 or EF1α) is the promoter from the Arabidopsis thalianaelongation factor gene 1α, AtEF1α-i (AtEF1α-i) is the intron in the 5′UTR of the Arabidopsis thaliana elongation factor gene 1α.

[0158] FIGS. 1-18 provide examples of plant transformation constructsthat contain one to three plant expression cassettes. Multiplecombinations of plant expression cassettes comprising the promoter andgenetic elements of the present invention can be constructed and testedin crops plants by those skilled in the art of plant molecular biologywithout undue experimentation. The constructs illustrated in the Figuresare not to be construed as the only constructs that can be assembled,but serve only as examples to those skilled in the art. FIG. 1(pCGN8086) provides an example of a plant transformation constructcontaining one expression cassette comprising one promoter of thepresent invention (P-AtAct8) operably linked to a gene of interest(CTP2-aroA:CP4syn). FIG. 2 (pMON45325) provides an example of a planttransformation construct containing two expression cassettes comprisingat least one promoter of the present invention (P-AtAct11) operablylinked to at least one gene of interest (CTP2-aroA:CP4syn). FIG. 3(pMON4533 1) provides an example of a plant transformation constructcontaining one expression cassette comprising one promoter of thepresent invention (P-AtEF 1 plus intron) operably linked to at least onegene of interest (CTP2-aroA:CP4syn). FIG. 4 (pMON45332) provides anexample of a plant transformation construct containing two expressioncassettes comprising at least one promoter of the present invention(P-AtEF1 plus intron) operably linked to at least one gene of interest(CTP2-aroA:CP4syn). FIG. 5 (pMON9190) provides an example of a planttransformation construct containing three expression cassettes whereinat least two promoters of the present invention (P-ATEF1 plus intron,AtEF1α-i; P-AtAct2 plus intron, AtAct2i) are operably linked to at leastone gene of interest (CTP2-aroA:CP4syn) and the P-eFMV promoter operablylined to CTP2-aroA:CP4syn. FIG. 6 (pMON9153) plant expression cassettesare identical to those illustrated in FIG. 4 (pMON45332), this plasmidmap is illustrated for the purpose of identification of the expressioncassettes for data shown on plant phenotype in the data tables shown inthe specification. FIG. 7 (pCGN8099) provides an example of a planttransformation construct containing two expression cassettes comprisinghybrid promoters of the present invention, P-eFMV-AtEF1α andP-e35S-AtAct8, driving transcription of the gene of interest(aroA:CP4syn). FIG. 8 (pCGN8088) provides an example of a planttransformation construct containing two expression cassettes comprisingone promoter of the present invention, P-AtAct8 plus intron, AtAct8i,and the P-eFMV promoter driving expression of a gene of interest(aroA:CP4syn). FIG. 9 (pCGN8068) provides an example of a planttransformation construct containing two expression cassettes comprisingone promoter of the present invention, P-AtAct2 plus intron, AtAct2i,and the P-eFMV promoter driving expression of a gene of interest(aroA:CP4syn). FIG. 10 (pCGN8096) provides an example of a planttransformation construct containing two expression cassettes comprisinghybrid promoters of the present invention, P-eFMV/-AtAct11 andP-e35S-AtAct2, driving transcription of the gene of interest(aroA:CP4syn). FIG. 11 (pCGN9151) provides an example of a planttransformation construct containing two expression cassettes comprisinghybrid promoters of the present invention, P-eFMV-AtEF1α andP-e35S-AtAct2, driving transcription of the gene of interest(aroA:CP4syn). FIG. 12 (pMON10156) provides an example of a planttransformation construct containing one expression cassette comprisingthe P-eFMV promoter driving expression of the aroA:CP4syn gene ofinterest, this vector is used for comparative purposes with the promotersequences of the present invention. FIG. 13 (pMON52059) provides anexample of a plant transformation construct containing one expressioncassette comprising a hybrid promoter (P-eFMV-AtEF1α) driving theexpression of the gene of interest (aroA:CP4syn). FIG. 14 (pMON54952)provides an example of a plant transformation construct containing oneexpression cassette comprising one promoter of the present invention(P-AtAct1a plus AtAct1a intron) operably linked to at least one gene ofinterest (CTP2-aroA:CP4syn). FIG. 15 (pMON54953) provides an example ofa plant transformation construct containing one expression cassettecomprising one promoter of the present invention (P-AtAct1b plus AtAct1bintron) operably linked to at least one gene of interest(CTP2-aroA-CP4syn). FIG. 16 (pMON54954) provides an example of a planttransformation construct containing one expression cassette comprisingone promoter of the present invention (P-AtAct3 plus AtAct3 intron)operably linked to at least one gene of interest (CTP2-aroA:CP4syn).FIG. 17 (pMON54955) provides an example of a plant transformationconstruct containing one expression cassette comprising one promoter ofthe present invention (P-AtAct7 plus AtAct7 intron) operably linked toat least one gene of interest (CTP2-aroA:CP4syn). FIG. 18 (pMON54956)provides an example of a plant transformation construct containing oneexpression cassette comprising one promoter of the present invention(P-AtAct12 plus AtAct12 intron) operably linked to at least one gene ofinterest (CTP2-aroA:CP4syn).

Example 2

[0159] The cloning constructs and GUS constructs are listed in Table 1.The Arabidopsis actin 2 promoter and intron (Genbank accession numberU41998 as described in An et al., Plant J. 10: 107-121, 1996) wasisolated using Arabidopsis thaliana Landsberg erecta DNA as a template(Rogers and Bendich, Plant Mol. Biol. 5:69, 1998) using SEQ ID NO:1(forward primer) and SEQ ID NO:2 (reverse primer) in a reaction asfollows: 0.5 μg template DNA, 25 pmole of each primer, taq polymerase(BMB, Indianapolis, Ind.) using wax beads for “hot start” PCR. The PCRthermocycler conditions were as follows: 94° C. for one minute; 30cycles of: 92° C. for 40 seconds, 55° C. for one minute, 72° C. for oneminute and 30 seconds; and a five minute 72° C. extension. The PCRreaction was purified using GeneClean II (Bio101 Inc., Vista, Calif.),digested with HindIII and NcoI, and ligated into construct pMON26149(Table 1) digested with HindIII and NcoI. The promoter clone wassequence verified and the resulting construct was designated pMON26170(Table 1). TABLE 1 Cloning Constructs and GUS Constructs containingArabidopis Actin and EF1 promoter sequences Construct DescriptionPromoter*/Gene/3′ pMON26149 cloning construct pMON26170 plant expressionconstruct Act2/GUS/nos pMON26171 plant expression construct Act8/GUS/nospMON8677 cloning construct pMON48407 plant expression constructAct11/GUS/nos pMON26152 cloning construct pMON26177 plant expressionconstruct EF1/GUS/nos pMON11750 plant expression construct e35S/GUS/nospMON15737 plant expression construct FMV/GUS/nos

Example 3

[0160] The Arabidopsis actin 8 promoter and intron (Genbank accessionnumber U42007 as described in An et al., Plant J. 10:107-121, 1996) wasisolated using Arabidopsis thaliana Landsberg erecta DNA as a templatePCR conditions and purification methods described in Example 2 usingprimers SEQ ID NO:3 (forward primer) and SEQ ID NO:4 (reverse primer).The promoter was cloned using restriction enzymes as described inExample 2, sequence verified, and the resulting construct was designatedpMON26171 (Table 1).

Example 4

[0161] The Arabidopsis actin 11 promoter and intron (Genbank accessionnumber U27981 as described in Huang et al., Plant Mol. Biol.,33:125-139, 1997) was isolated using Arabidopsis thaliana Landsbergerecta DNA as a template PCR conditions and purification methodsdescribed in Example 2 using primers SEQ ID NO:5 (forward primer) andSEQ ID NO:6 (reverse primer). The promoter was cloned using restrictionenzymes EcoRV and NcoI and ligated into pMON8677 (Table 1), sequenceverified, and the resulting construct was designated pMON48407 (Table1).

Example 5

[0162] The Arabidopsis elongation factor 1α (AtEF1α) promoter and intron(Genbank accession number X16430 as described in Axelos et al., Mol.Gen. Genet. 219:106-112, 1989; Curie et al., NAR 19:1305-1310; Curie etal., Plant Mol. Biol. 18:1083-1089, 1992; Curie et al., Mol Gen. Genet.238:428-436, 1993) was isolated using Arabidopsis thaliana Landsbergerecta DNA as a template PCR conditions and purification methodsdescribed in Example 2 using primers SEQ ID NO:7 (forward primer) andSEQ ID NO:8 (reverse primer). The promoter was cloned using restrictionenzymes HindIII and NcoI and ligated into pMON26152 (Table 1) asdescribed in Example 2, sequence verified, and the resulting constructwas designated pMON26177 (Table 1).

Example 6

[0163] The plant transformation constructs described were mated intoAgrobacterium. Cotton transformation was performed essentially asdescribed in WO/0036911, herein incorporated by reference in itsentirety. The Arabidopsis transformation was performed as described inYe et al., Plant Journal 19:249-257, 1999. The tomato transformation wasperformed as described in U.S. Pat. No. 5,565,347 herein incorporated byreference in its entirety.

Example 7

[0164] A DNA construct is transformed into a target crop of interest viaan appropriate delivery system such as an Agrobacterium-mediatedtransformation method (see for example U.S. Pat. No. 5,569,834 hereinincorporated by reference in its entirety, U.S. Pat. No. 5,416,011herein incorporated by reference in its entirety, U.S. Pat. No.5,631,152 herein incorporated by reference in its entirety, U.S. Pat.No. 5,159,135 herein incorporated by reference in its entirety, U.S.Pat. No. 5,004,863 herein incorporated by reference in its entirety, andU.S. Provisional Appln. No. 60/111795 herein incorporated by referencein its entirety. Alternatively, a particle bombardment method may beused (see for example Patent Applns. WO 92/15675. WO 97/48814 andEuropean Patent Appln. 586,355, and U.S. Pat. Nos. 5,120,657, 5,503,998,5,830,728 and 5,015,580, all of which are herein incorporated byreference in their entirety).

[0165] A large number of transformation and regeneration systems andmethods are available and well-known to those of skill in the art. Thestably transformed plants and progeny are subsequently analyzed forexpression of the gene in tissues of interest by any number ofmolecular, immunodiagnostic, biochemical, and/or field evaluationmethods known to those of skill in the art, including, but not limitedto a spray test with a glyphosate formulation at commercially effectiveconcentrations performed in a growth chamber or field environment.

Example 8

[0166] The GUS assays are performed by routine methods known to those ofskill in the art (see for example, Jefferson et al., EMBO J. 6:3901,1987). For cotton, R0 plants were tested. The tissue was size selectedat various stages in development, samples and pooled for analysis. Thecotton floral bud was harvested and the male reproductive tissue samples(anthers and filaments), female reproductive tissue samples (entirestigma, style, and ovary), and corolla (sepals and petals) were taken.For the size selection, three floral buds from each stage were selectedthat included several sizes including small (less than 0.5 cm), medium(from 0.5-0.7 cm), and large (candle stage or open flower). Leaf sampleswere collected about 1-2 weeks after the cotton plants were placed inthe greenhouse, and the other samples were collected approximately 1-2months later. The first flowers were not collected (the first fivefruiting positions were left intact).

[0167] For Arabidopsis, V1 plants were analyzed and only homozygous andheterozygous segregants were tested Eight to ten events per constructwere analyzed (five plants per event). The GUS results for Arabidopsisrepresent pooled samples of 8- 10 events. The values in the disclosedtables (Table 2 and Table 3) represent the average GUS expression forthe designated tissue (pmol/MU/min/mg).

Example 9

[0168] Plants were analyzed for GUS expression in leaf tissue andreproductive tissues including immature floral buds and flowers. Theresults are shown in Table 2. Constructs tested included pMON48407(P-AtAct11+intron/GUS/nos), pMON26170 (P-AtAct2+intron/GUS/nos),pMON26171 (P-AtAct8+intron/GUS/nos), pMON11750 (e35S/GUS/nos), pMON26177(P-EF1α+intron/GUS/nos), and pMON15737 (P-FMV/GUS/nos). The actin andelongation factor promoters conferred high levels of GUS expression inmultiple tissues including reproductive tissues. TABLE 2 AverageArabidopsis V1 GUS Expression Immature Construct Leaf Floral Bud FlowerGynoecium Andorecium pMON48407 6944 7394 8359 ND ND pMON26170 4523874099 54502 73623 217292 pMON26171 29343 35884 37125 76311 207100pMON11750 60844 14032 16263 35882 115049 pMON26177 47598 72871 96420191066 507370 pMON15737 28314 57903 84457 44696 87876

Example 10

[0169] The R0 cotton plants were tested for expression of the GUSreporter gene in selected tissues of various stages of development. Thefloral buds were staged by size (small, medium, and large; large=candleand open flower). The androecium represented the male reproductivetissues including the entire receptacle (stigma, style, and ovaries).The corolla sample was composed of sepals and petals. The tissue wasprepared and GUS assays performed as described in EXAMPLE 8. The resultsare summarized in Table 3. The constructs tested included pMON48407(P-EF1α+intron/gus/nos), pMON26170 (P-AtAct2+intron/gus/nos), andpMON48407 (P-AtAct11+intron/gus/nos).

[0170] Six plants were tested and average GUS values obtained forpMON26177. Twenty plants were tested and average GUS values obtained forfor pMON26170. Eight plants were tested and average GUS values obtainedfor pMON48407. The results demonstrate that the actin and elongationfactor promoters can be used for effective expression of operably linkedgenes, particularly in reproductive tissues TABLE 3 GUS Assay Resultsfor Cotton Plants Construct Promoter/intron Tissue Tested GUS ResultspMON26177 EF1α Leaf 11600 pMON26177 EF1α Small Corolla  396 pMON26177EF1α Small Gynoecium  8670 pMON26177 EF1α Small Androecium 13771pMON26177 EF1α Medium Corolla  362 pMON26177 EF1α Medium Gynoecium  3318pMON26177 EF1α Medium Androecium  8006 pMON26177 EF1α Large Corolla  351pMON26177 EF1α Large Gynoecium  500 pMON26177 EF1α Large Androecium15512 pMON26170 Act2 Leaf 12718 pMON26170 Act2 Small Corolla  1296pMON26170 Act2 Small Gynoecium 16684 pMON26170 Act2 Small Androecium 7570 pMON26170 Act2 Medium Corolla  742 pMON26170 Act2 Medium Gynoecium10041 pMON26170 Act2 Medium Androecium  7893 pMON26170 Act2 LargeCorolla  289 pMON26170 Act2 Large Gynoecium  3218 pMON26170 Act2 LargeAndroecium 42737 pMON48407 Act11 Leaf 28289 pMON48407 Act11 SmallCorolla   10 pMON48407 Act11 Small Gynoecium 40755 pMON48407 Act11 SmallAndroecium 47834 pMON48407 Act11 Medium Corolla  742 pMON48407 Act11Medium Gynoecium 52495 pMON48407 Act11 Medium Androecium 35573 pMON48407Act11 Large Corolla  1072 pMON48407 Act11 Large Gynoecium  4869pMON48407 Act11 Large Androecium 42737

Example 11

[0171] Transformed plants were also tested in a greenhouse spray testusing Roundup Ultra™ a glyphosate formulation with a Track Sprayerdevice (Roundup Ultra is a registered trademark of Monsanto Company).Plants were at the “two” true leaf or greater stage of growth and theleaves were dry before applying the Roundup® spray. The formulation usedwas Roundup Ultra™ as a 3 lb/gallon a.e. (acid equivalent) formulation.The calibration used was as follows:

[0172] For a 20 gallons/Acre spray volume: Nozzle speed: 9501 evenflowSpray pressure: 40 psi Spray height 18 inches between top of canopy andnozzle tip Track Speed 1.1 ft/sec., corresponding to a reading of 1950—1.0 volts. Formulation: Roundup Ultra ™ (3 lbs. A.e./gallon)

[0173] The spray concentrations will vary, depending on the desiredtesting ranges. For example, for a desired rate of 8 oz/acre a workingsolution of 3.1 ml/L is used, and for a desired rate of 64 oz/A aworking range of 24.8 ml/L is used.

[0174] The evaluation period will vary, depending on the crop, stage ofplant development, and tolerance level desired.

Example 12

[0175] The plant expression constructs used for tomato transformationare listed in Table 4. Tomato plants (T0) containing constructscomprising at least one actin or elongation factor promoter (withintron) operably linked to an aroA:CP4 glyphosate tolerance gene arescreened in a greenhouse glyphosate spray test with glyphosate (RoundupUltra™) formulation for the efficiency of conferring glyphosatetolerance to transgenic tomato plants. Optionally, at least one actin orelongation factor promoter sequence operably linked to an aroA:CP4 geneand an eFMV caulimovirus promoter operably linked to an aroA:CP4transformed into tomato plants are screened by spray application withglyphosate (Roundup Ultra™). Tomato plants are sprayed with 48 oz./acrethen evaluated at two weeks post application for analysis of vegetativetolerance and up to 60 days post-application for analysis ofreproductive tolerance. The results are shown in Table 4 and rankedaccording to efficiency of selecting reproductive tolerant lines. Thepercent vegetative tolerance is the percentage of the lines screenedthat demonstrated sufficient vegetative tolerance to glyphosate damageto be considered for further studies of agronomic traits in preparationfor commercially candidacy. The percent reproductive tolerance is thepercentage of the vegetative tolerant lines that also demonstratedsufficient reproductive tolerance to be considered for further agronomicevaluation. All of the constructs proved functional for providingvegetative tolerance and reproductive tolerance to the transgenic tomatoplants. Various combinations of promoters are able to increase theefficiency at which vegetative and reproductive tolerant lines could beselected by screening in this experiment. Constructs containing theArabidopsis EF1α promoter are more specifically associated with a highpercentage of vegetatively tolerant lines. P-Act2 promoter incombination with P-eFMV and P-AtEF1α (pCGN9190) provided an increase inthe percentage of reproductively tolerant lines that are screened bythis method. TABLE 4 Greenhouse Track Spray Trials with Application Rateof 48 oz./Acre* # Lines % Vegetative % Reprod. Construct DescriptionTested Tolerance¹ Toler.² pCGN9190 eFMV/CP4 + 930 83.2 52.4 EF1α/CP4 +Act2/CP4 pCGN9153 EF1α/CP4 + 391 73.9 38.9 eFMV/CP4 pCGN8086 Act8/CP4 21 47.6 38.1 pCGN8099 EF1α/CP4 +  71 84.5 36.6 Act8/CP4 pCGN8088eFMV/CP4 + 144 79.9 34.7 Act8/CP4 pMON45325 eFMV/CP4 +  90 70.0 34.4Act11/CP4 pCGN8096 Act11/CP4 + 201 62.7 10.4 Act2/CP4 pCGN8067 Act2/CP4205 67.3  8.8

[0176] Tomato seed yield is used as a measure of the efficacy of thevarious promoter sequences and combination of expression cassettes usedin the present invention for conferring glyphosate tolerance totransgenic tomato plants. In Table 5, the results of three fieldexperiments are shown on transgenic tomato plants containing constructswith the promoters of the present invention driving expression of thearoA:CP4 coding sequence for glyphosate tolerance. Experiment I is atest of the plants produced from the constructs that contain the Figwortmosaic virus promoter (P-FMV) in the native and the duplicated enhancedversion (P-eFMV) and additional genetic elements in the constructs thatare also found in the constructs used to test the promoter sequences ofthe present invention. Additional genetic elements such as the source ofthe 5′ untranslated sequence and the chloroplast transit peptide arealso tested. The construct pMON20998 comprises the P-eFMV, linked to thepetunia Hsp70 5′ UTR, leader linked to the Arabidopsis EPSPS chloroplasttransit peptide (CTP2), linked to the E9 3′ termination region. Theconstruct pMON20999 differs from pMON20998 only in that the promoter isP-FMV. The construct pMON10156 differs from pMON20998 only in that theCTP is from the Petunia EPSPS chloroplast transit peptide (CTP4). Theconstruct pMON45312 differs from pMON20998 only in that the leadersequence is the native FMV leader sequence.

[0177] Tomato plants are transplanted into the field in rows. The plantsare spray treated in the field at a rate of 48 oz./Acre with Roundupherbicide. The tomato seed is collected from the fruit and weighted. Anunsprayed tomato line serves as the control for comparison purposes andthe efficacy of each construct is expressed as a percentage of thecontrol. The result of Experiment 1 (column 1 of Table 5) is that theFMV promoter and P-eFMV only provide 5-11 % of the seed production of anunsprayed check. Experiment 2, and 3 tests the constructs of the presentinvention at different locations (columns 2 and 3 of Table 5).Experiment 2 is conducted at the same location as Experiment 1, theconstructs pCGN8099 (FIG. 7), pCGN9151 (FIG. 11) and pCGN9190 (FIG. 5)performed well by providing 25-46% of the seed relative to an unsprayedcheck. At a different location that has a cooler growing season,Experiment 3 demonstrated that pCGN8068 (FIG. 9), pCGN8088 (FIG. 8),pCGN8099, pCGN9151, pCGN9153 (FIG. 6), and pMON45325 (FIG. 2) are ableto confer sufficient glyphosate tolerance for the tomatoes to set 34-77%of normal seed set relative to an unsprayed check. TABLE 5 Tomato seedyield experiments Exp. 1 Exp. 2 Exp. 3 Seed wt % of Seed wt % of Seed wt% of grams Control grams Control grams Control pMON20998 0.52  5.3pMON20999 0.84  8.6 pMON10156 0.50  5.1 pMON45312 1.07 11.0 pCGN80680.48  8.4 7.06 77.8 pCGN8088 0.43  7.6 3.09 34.1 pCGN8096 0.40  7.0pCGN8099 1.85 32.5 6.93 76.4 pCGN9151 1.46 25.7 6.11 67.4 pCGN9153 0.6812.0 4.03 44.4 pCGN9190 2.64 46.4 pMON45325 0.31  5.4 3.37 37.2 pCGN8067Control 9.73 100.0  5.69 100.0  9.07 100.0 

Example 13

[0178] SEQ ID NOS: 1-8, and SEQ ID NOS: 13-21 are PCR primers designedfrom publicly available sequence information for Arabidopsis thalianaAct 1, Act2 (Genbank #U41998), Act3, Act7, Act8 (Genbank #ATU42007),Act11 (Genbank #ATU2798 1), Act12 and Elf1α (Genbank #X16430) genes.These sequences are used to extend the nucleic acid sequence usingpolymerase chain reaction (PCR) amplification techniques (see forexample, Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263,1986; Erlich, et al., European Patent Appln. 50,424; European PatentAppln. 84,796, European Patent Appln. 258,017, European Patent Appln.237,362; Mullis, European Patent Appln. 201,184; Mullis, et al., U.S.Pat. No. 4,683,202; Erlich, U.S. Pat. No. 4,582,788; and Saiki, et al.,U.S. Pat. No. 4,683,194). A number of PCR amplification methods areknown to those of skill in the art and are used to identify nucleic acidsequences adjacent to a known sequence. For example, inverse PCR (IPCR)methods, which are used to amplify unknown DNA sequences adjacent to acore region of known sequence have been described. Other methods arealso available such as capture PCR (Lagerstrom M., et al., PCR MethodsApplic. 1:111, 1991), and walking PCR (Parker, et al., Nucleic Acids Res19:3055, 1991). A number of manufacturers have also developed kits basedon modifications of these methods for the purposes of identifyingsequences of interest. Technical advances including improvements inprimer and adaptor design, improvements in the polymerase enzyme, andthermocycler capabilies have facilitated quicker, efficient methods forisolating sequences of interest. TABLE 5 Primer sequences for isolationof Arabidopsis Actin and EF1α promoter sequences At. Actin 2 forward:TTTTTTTTGATATCAAGCTTCAACTATFITJ7ATGTATGC At. Actin 2 reverse:GCCTCAGCCATGGTGAGTCTGCTGCAAACACACAAAAAGAGTTCAAT At. Actin 8 forward:TTTTTTTTGATATCAAGCTTCCATTTTTCT TTTGCATAAT TC At. Actin 5 reverse:GCATCGGCCATGGTGAGTCTTCTGCAATCAAAAACATAAAGATCTGA At. Actin 11 forward:TTTTTTTTTAAGCTTGATATCACAACCAAATGTCAAATGG At. Actin 11 reverse:CCATCTGCCATGGTCTATATCCTGTC At. EF1α forward:TTTTTTTTTAAGCTTGATATCGGAAGTTTCTCTCTTG At. EF1α reverse:CTTTTCCCATGGTAGATCTCTGGTCAACAA ATC At. Actin 1a forward:CCCAAGCTTAAATGACATCAGATACACGC At. Actin 1b forward:CATAAGCTTAGAGGTCCAAATTCA At. Actin 1 reverse:CCATCAGCCATGGTCTTCTACCTTTATGCAAA At. Actin 3 forward:CCAAGCTTACCACACTCAGATGCATAAACAAACACA At. Actin 3 reverse:CATCAGCCATGGTCTACTCTCTGCAAAAACA At. Actin 7 forward:GCAAAGCTTACTAGTCAACAATTGGCC At. Actin 7 reverse:GATCGGCCATGGTTCACTAAAAAAAAAG At. Actin 12 forward:GGAAGCTTGCGGCCGCTTTCTACTCTACATGTTTCT At. Actin 12 reverse:GACTAGCCGCCATGGTTCAATCTCTAGCTGA

[0179] The leaves of young plants of Arabidopsis thaliana (1 g) werehomogenized in 9 ml of CTAB buffer (Saghai-Maroof et al. 1984, PNAS81:8014-8018). The CTAB buffer contained 100 mM TrisHCl, pH 7.8, 700 mMNaCl, 50 mM EDTA, 1% CTAB (alkytrimethyhylammoniumbromide) and 140 mM2-mercaptoethanol. After 90 minutes incubation in 65 C, 4.5 ml ofchloroform:isoamyl alcohol (24: 1) was added and samples were mixed for10 minutes. Aqueous layer was separated by centrifugation for 10 minutesat 1500 g and was re-extracted with chloroform:isoamyl alcohol. Aftersecond centrifugation, aqueous layer was transferred to a tubecontaining 50 μl 10mg/ml RNase A (DNase free) and incubated in roomtemperature for 30 minutes to remove RNA. DNA was precipitated with 6 mlof isopropanol and re-suspended in 1 ml of 10 mM TrisHCl buffer pH 8.5.DNA solution was extracted once with equal volume of phenol and oncewith an equal volume of chloroform: isoamylalcohol. Aftercentrifugation, {fraction (1/10)} volume of sodium acetate (3M, pH 5.2)was added to aqueous layer, followed by 2.5 volume of ethanol. The DNAwas hooked, washed in 70% ethanol, then air dried and re-suspended in0.2 ml of 10 mM TrisHCl buffer.

[0180] Arabidopsis genomic DNA (100 ng) was used in 50 μl PCR reactions.Reactions containing the primers shown in Table 5. contained 10 μMreverse and forward primer solutions, 200 nM dNTPs and PCR buffer withmagnesium and DNA polymerase mix from Expand™ High Fidelity PCR System(Roche Molecular Biochemicals). After initial 2 minute denaturation at94° C. reactions were cycled 0.5 min at 94° C., 0.5 min at 55° C. and1.5 minute at 72° C. for 35 times. PCR products were analyzed byelectrophoresis on 1% agarose gel. Gel isolated DNA fragmentsrepresenting Actin 1a, Actin 1b, Actin 7, and Actin 12 sequences werephosphorylated with T4 DNA kinase and ligated to dephosphorylated andSma I cut pUC19 cloning construct. White colonies were screened for thepresence of appropriate inserts and sequenced with M13 forward andreverse primers to confirm the presence of actin promoters. Selectedclones were designated as pMON54941 (P-AtAct1a), pMON54942(P-AtAct1b),pMON54943 (P-AtAct7) and pMON54944 (P-AtAct12). Subsequently, the Actinpromoters DNA fragments were released by Hind III and NcoI digest of thepUC19 constructs containing the insert sequences, the DNA fragments weregel isolated and ligated to pMON26165 that had been digested with thesame restriction enzymes. A PCR product for the Actin 3 promoter(P-AtAct3) was digested with Hind III and Nco I and cloned directly intopMON26165 to form pMON5495 1. pMON26165 contains the GUS/nos terminatorgene segment. Ligation with the promoter segments allows for assay ofeach promoter for functional activity by expression of theβ-glucuronidase enzyme in plant cells. The plant cells can be isolated,for example, tobacco leaf protoplasts, or the plant cells may becontained in a plant tissue or organ, such as, leaf, root, cotyledon,hypocotyl, embryo, flower, or storage organ.The expression level of GUSdriven by these promoters is assayed in soybean hypocotyl in comparisonwith GUS driven by P-e35S promoter (Table 6). Plasmid DNA/gold particleswas bombarded to soybean hypocotyls then after 48 hours the GUS activitywas assayed histochemically. All of the Actin promoters tested in thisassay show functional activity in the hypocotyl tissue demonstratingtheir utility for expression transgenes in heterologous crop plantspecies.

[0181] The constructs containing aroA:CP4 EPSPS driven by theArabidopsis Actin 1a, (pMON54952), Actin 1b (pMON54953), Actin 3(pMON54954), Actin 7 (pMON54955) and Actin 12 (pMON54956) promoters ofthe present invention were prepared in Agrobacterium binary planttransformation constructs for stable expression of the glyphosateresistant EPSPS in crop plants. These constructs are transformed intosoybean and cotton cells, the cells are selected and regenerated intoplants on glyphosate containing tissue culture media and then assayedfor expression of the aroA:CP4 protein and for tolerance to glyphosateapplication. Plants demonstrating commercially acceptable glyphosatetolerance are further developed by conventional breeding methods totransfer the glyphosate tolerance trait into germplasm adapted forcultivation. TABLE 6 Activity of different Arabidopsis actin promotersin transient assay as compare to P-e35S. Construct GUS ActivityPe35S/GUS +++ P-AtAct1a/GUS ++ P-AtAct1b/GUS ++ P-AtAct3/GUS ++P-AtAct7/GUS ++ P-AtAct12/GUS +

Example 14

[0182] Cotton yield is correlated with the number of squares set duringthe first four to five weeks of squaring. The retention of these squaresto mature bolls and their contribution to the harvest of the cotton lintis a key component of yield. When determining the efficacy of transgeneconstructs for conferring herbicide tolerance in cotton, the amount ofboll retention is a measure of efficacy and is a desirable trait.Transgenic cotton plants containing promoters of the present invention(Table 7) were assayed in greenhouse conditions for boll retention. Thepromoters directed expression of the aroA:CP4 coding sequence forglyphosate tolerant phenotype. The plants were transformed by anAgrobacterium-mediated method or by a particle gun method. The particlegun constructs contained an additional GUS containing expressioncassette useful for histochemical localization of β-glucuronidaseactivity from the promoters of the present invention. Transgenic plantswere regenerated on glyphosate containing media and plants rooted on arooting media. The rooted plantlets were potted in soil and transferredto a growth chamber for a hardening off period. The seed from theseplant lines were collected and planted. Fifteen plants from each linewere sprayed with glyphosate at 48 ounces/acre at the 4 leaf stage. Atleast 8 surviving plants from each line were sprayed again at the 8 leafstage with glyphosate at 48 ounces/acre. At maturity, the number offirst position bolls for the first five bolls was counted. Those linesthat had 3 or more of the first position bolls retained after theglyphosate spray (plant map≧3) were advanced for further study. Table 7illustrates the data produced from this study. The number of linesmapped indicates the number of lines surviving the first glyphosatespray application. The commercial standard is Line 1445 (pMON17136) thatcontains the P-FMV promoter driving expression of the CTP2-aroA:CP4gene/E9 3′, this line retains less than 1 of the 5 first bolls. Theconstructs, pCGN8099, pCGN9153, pCGN8088, pCGN8068 provided sufficientreproductive glyphosate tolerance in cotton such that 14-35% of thelines tested from these constructs were advanced for further agronomictrials. TABLE 7 Greenhouse cotton boll retention study # lines PlantConstruct Promoters Mapped Map ≧ 3 % ≧ 3 pCGN8099 eFMV:EF1a + e35S:Act8104  36 34.6% pCGN9153 EF1α + FMV 36 12 33.3% pCGN9165 EF1α + 35S/GUS  31 33.3% pCGN9152 EF1a  7 0  0.0% pCGN8088 Act8 + FMV 43 6 14.0% pCGN8086Act8  7 0  0.0% pCGN8068 Act2 + FMV 37 7 18.9% pCGN8067 Act2 37 0  0.0%pCGN8084 Act2 + FMV + 35S/GUS  5 0  0.0% pCGN8085 Act2 + FMV/GUS  1 0 0.0% pCGN9164 Act11 + 35S/GUS 21 1  4.8% pMON45325 Act11 + FMV 43 0 0.0% pCGN8096 eFMV:Act11 + e35S:Act2 14 0  0.0% pCGN9154 eFMV:Act11 +e35S:Act2 16 1  6.3% Line 1445 FMV <1.0

Example 15

[0183] Cotton yield is correlated with the number of squares set duringthe first four to five weeks of squaring. The retention of these squaresto mature bolls and their contribution to the harvest of the cotton lintis a key component of yield. When determining the efficacy of transgeneconstructs for conferring herbicide tolerance in cotton, the amount ofboll retention is a measure of efficacy and is a desirable trait.Transgenic cotton plants containing promoters of the present inventionwere assayed in field conditions at two locations for boll retention.The transgenic cotton lines 502-254-2 (pCGN8068), 701-178-2 (pCGN8068),53-2 (pCGN8088), 178-1 (pCGN9153), and 60-1 (pCGN9153) were compared to1445 (glyphosate tolerance line) and PM1218BR (Paymaster 1218 parent)that contain the construct pMON17136 (P-FMV/CTP2-aroA:CP4/E93′), a wildtype non-transgenic line, Coker 130 was included. The field design is arandomized complete block design consisting of 2 rows×20-30 feet×3replications. Glyphosate is applied as Roundup Ultra™ formulation atrates of 1.12 lb ai/A=48 oz product and 1.5 lb ai/A=64 oz product at the8 leaf stage of cotton plant development. All of the cotton plots aremanaged aggressively for weed and insect pest control, as well as otheragronomic inputs such as planting time, fertilization, irrigation, PGRusage and defoliation. The percent boll retention is determined bymapping the location of each of the retained bolls by random selectionof ten plants from the middle of the two center rows (five from eachrow) of each plot to map. The first mapping should be done 4 weeks afterfirst flower (mid-season map), a second mapping should be done atharvest. The data collected includes the number of first position bollson the bottom five flowering nodes that are counted as an indication ofthe reproductive tolerance of the transgenic cotton lines to glyphosate.Table 8 illustrates the advantage that promoters of the presentinvention have conferred to transgenic cotton plants for boll retention.This enhanced reproductive tolerance has resulted in increased lintyield (Table 9) and increased seed yield (Table 10) as well. TABLE 8Boll retention at mid-season plant map of bottom 5 first position boilsLocation 1 Location 2 48 64 48 64 Untreated oz/A oz/A Untreated oz/Aoz/A (17136) 1445 68 67 53 81 63 62 (8068) 502-254-2 87 72 64 77 80 69(8068) 701-178-2 85 77 60 84 86 76 (8088) 53-2 89 81 80 79 76 73 (9153)178-1 77 83 73 85 71 79 (9153) 60-1 80 89 81 77 82 87 PM1218BR 92 56 63

[0184] TABLE 9 Lint Yield (lbs/Acre) and percent yield (Location 1)Cultivar Untreated 48 oz/A 48 oz/A % 64 oz/A 64 oz/A % 8068-502-254-2-41103 960  87.0% 858 77.8% 8068-701-178-2-2 1326 1219  91.9% 1177 88.8%9153-60-1-1 1177 1206 102.5% 1171 99.5% 9153-178-1-1 1112 769  69.2% 75067.4% 8088-53-2-11 1283 1071  83.5% 1097 85.5% 1445 1018 563  55.3% 49048.1% C130 1200 0  0.0% 0  0.0% PM 1218 BR 1092 826  75.6% 713 65.3%

[0185] TABLE 10 Seed Cotton Yield (lbs/Acre) and percent yield(Location 1) Cultivar Untreated 48 oz/A 48 oz/A % 64 oz/A 64 oz/A %8068-502-254-2-4 3357 2923  87.1% 2646  78.8% 8068-701-178-2-2 3720 3521 94.7% 3328  89.5% 9153-60-1-1 3294 3413 103.6% 3316 100.7% 9153-178-1-13468 2355  67.9% 2218  64.0% 8088-53-2-11 3404 2950  86.7% 2968  87.2%1445 2835 1624  57.3% 1372  48.4% C130 3272 0  0.0% 0  0.0% PM 1218 B/RR3036 2192  72.2% 1885  62.1%

Example 16

[0186] The efficacy of the hybrid promoter P-eFMV-AtEF a drivingexpression of the CTP2-aroA:CP4 coding sequence (FIG. 13, pMON52059) andP-FMV/CTP2-aroA:CP4/E93′ (pMON15737) was compared in transgenicArabidopsis thaliana. The transgenic Arabidopsis thaliana plants wereproduced by the vacuum infiltration (Bechtold et al., C R Acad ParisLife Sci 316: 1194-1199) seeds were potted in soil in trays in a growthchamber adjusted for 24° C., 16 hour light (120 μE m⁻²s⁻¹) cycle topermit normal growth and development of the plants. The pMON52059 V1event glyphosate tolerant transgenic Arabidopsis plants were selected byspray application of glyphosate herbicide at a rate of 24 ounces/acre,the surviving plants were transplanted into individual pots. EightpMON52059 V1 plants and eight pMON15737 homozygous plants were sprayed asecond time corresponding to the observation of bolting, approximately16 days after the at a rate of 24 ounces/acre. The second spray willdetermine the efficacy of the two constructs for conferring reproductivetolerance. The plants were observed for vegetative effects of glyphosateapplication. All plants had complete vegetative tolerance and noabnormal flowers were observed. However, abortion of siliques occurredindicated that seed had not been set in the aborted siliques. The totalnumber of siliques produced by each plant and the siliques thatcontained seeds (fertile siliques) were counted and tabulated. Theresults are shown in Table 9 and indicate that the hybrid promoterconstruct pMON52059 demonstrated a greater than 10 fold improvement infertile siliques, 89% compared to pMON15737 at 8%. The number of fertilefruiting structures is related to the amount of seed that can beproduced, this is especially important in crops whose yield isassociated with seed numbers. Crops such as cotton, soybean, canola,wheat, and corn are crops where reproductive tolerance to glyphosate isessential for good yield. TABLE 11 Comparison of the hybrid promoterP-eFMV-EF1α (pMON52059) and P-FMV (pMON15737) in conferring reproductivetolerance to glyphosate in Arabidopsis plants. pMON52059 pMON15737 PlantFertile Total Percent Plant Fertile Total Percent Number SiliguesSiligues Fertility Number Siligues Siligues Fertility 8819 39 50 78.0% 174 540 13.7% 8820 626 691 90.6% 2 23 600  3.8% 8821 507 561 90.4% 3 1470  0.2% 8822 0 69  0.0% 4 20 646  3.1% 8823 512 534 95.9% 5 43 717 6.0% 8827 326 354 92.1% 6 22 651  3.4% 8833 432 461 93.7% 7 178 86820.5% 8838 323 374 86.4% 8 40 520  7.7% Total 2765 3094 89.4% Total 4015012  8.0%

Example 19

[0187] Sunflower (Helianthus annuus L.) is a crop of agronomicimportance for oil and food. The constructs pMON45325 (FIG. 2),pMON45332 (FIG. 4), and pMON45331 (FIG. 3) of the present invention weretransformed into sunflower. Agrobacterium-mediated transformation ofsunflower has been reported (Schrammeijer et al., Plant Cell Reports, 9:55-60, 1990; EP 0 486 234. Methods known by those skilled in the art ofplant transformation with transgene expression constructs can includehypocotyls, apical meristems, protoplasm, and other sunflower tissues.Transgenic sunflower lines SFB250-27 contains pMON20999 (P-FMV/CTP2-aroA:CP4/E93′) expression cassette; SFB288-01, SFB295-09contain pMON45325(P-eFMV/CTP2-aroA:CP4/E93′:P-AtAct11+intron/CTP2-aroA:CP4/E93′);SFB289-01 contains pMON45332(P-AtEF1α+intron/CTP2-aroA:CP4/E93′:P-eFMV/CTP2-aroA:CP4/E93′);SFB303-08, SFB303-09, SFB303-11, and HA300B contain pMON45331(P-AtEF1α+intron/CTP2-aroA:CP4/E9). These lines are tested forglyphosate tolerance and are shown in Table 12.

[0188] The reproductive tolerance to glyphosate in sunflower can bemeasured as a function of the percent of normal heads, percent normalhead size and the pollen production. These plants are sprayed withGlyphosate at V-4 and V-8 leaf stages at 0, 32 oz/acre or 64 ounces/acrerate. The sunflower plants are assessed for vegetative tolerance toglyphosate. Vegetative tolerance is achieved at 32 and 64 oz/acre levelsof glyphosate spray at both V4 and V8 stages of plant development.

[0189] Vegetative glyphosate tolerant transgenic sunflower lines arescored for number of heads, percent normal heads, percent normal headsize, and percent normal pollen shed. These traits are scored in a fieldtest at one location. The tabulation of the head scores and pollenproduction is shown in Table 12. Lines selected from the constructs ofthe present invention show greater percent of normal heads, generallygreater percent normal head size and better pollen shed. TABLE 12Sunflower glyphosate resistance scores % normal % normal % pollen Line ## heads heads head size shed SFB250-27 28 29 75 36 SFB288-01 11 36 73 73SFB295-09 28 57 64 68 SFB289-01 13 38 92 38 SFB303-08 25 68 92 64SFB303-09 43 81 88 88 SFB305-11 45 71 84 100  HA300B 30 100  97 97non-trans  0  0  0  0 segregant

Example 18

[0190] Cis acting regulatory elements necessary for proper promoterregulation can be identified by a number of means. In one method,deletion analysis is carried out to remove regions of the promoter andthe resulting promoter fragments are assayed for promoter activity. DNAfragments are considered necessary for promoter regulation if theactivity of the truncated promoter is altered compared to the originalpromoter fragment. Through this deletion analysis, small regions of DNAcan be identified which are necessary for positive or negativeregulation of transcription. Promoter sequence motifs can also beidentified and novel promoters engineered to contain these cis elementsfor modulating expression of operably linked transcribable sequences.See for example U.S. Pat. No. 5,223,419, herein incorporated byreference in its entirety, U.S. Pat. No. 4,990,607 herein incorporatedby reference in its entirety, and U.S. Pat. No. 5,097,025 hereinincorporated by reference in its entirety.

[0191] An alternative approach is to look for similar sequences betweenpromoters with similar expression profiles. Promoters with overlappingpatterns of activity can have common regulatory mechanisms. Severalcomputer programs can be used to identify conserved, sequence motifsbetween promoters, including but not limited to MEME, SIGNAL SCAN, orGENE SCAN. These motifs can represent binding sites for transcriptionsfactors which act to regulate the promoters. Once the sequence motifsare identified, their function can be assayed. For example, the motifsequences can be deleted from the promoter to determine if the motif isnecessary for proper promoter function. Alternatively, the motif can beadded to a minimal promoter to test whether it is sufficient to activatetranscription. Suspected negative regulatory elements can be tested forsufficiency by adding to an active promoter and testing for a reductionin promoter activity. Some cis acting regulatory elements may requireother elements to function. Therefore, multiple elements can be testedin various combinations by any number of methods known to those of skillin the art.

[0192] Once functional promoter elements have been identified, promoterelements can be modified at the nucleotide level to affect proteinbinding. The modifications can cause either higher or lower affinitybinding which would affect the level of transcription from thatpromoter.

[0193] Promoter elements can act additively or synergistically to affectpromoter activity. In this regard, promoter elements from different 5′regulatory regions can be placed in tandem to obtain a promoter with adifferent spectrum of activity or different expression profile.Accordingly, combinations of promoter elements from heterologous sourcesor duplication of similar elements or the same element can confer ahigher level of expression of operably linked transcribable sequences.For example, a promoter element can be multimerized to increase levelsof expression specifically in the pattern affected by that promoterelement.

[0194] The technical methods needed for constructing expressionconstructs containing the novel engineered 5′ regulatory elements areknown to those of skill in the art. The engineered promoters are testedin expression constructs and tested transiently by operably linking thenovel promoters to a suitable reporter gene such as GUS and testing in atransient plant assay. The novel promoters are operably linked to one ormore genes of interest and incorporated into a plant transformationconstruct along with one or more additional regulatory elements andtransformed into a target plant of interest by a suitable DNA deliverysystem. The stably transformed plants and subsequent progeny areevaluated by any number of molecular, immunodiagnostic, biochemical,phenotypic, or field methods suitable for assessing the desiredcharacteristic(s).

[0195] Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

[0196] All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1 30 1 39 DNA artificial sequence misc_feature (1)..(39) fully syntheticsequence 1 ttttttttga tatcaagctt caactatttt tatgtatgc 39 2 47 DNAartificial sequence misc_feature (1)..(47) fully synthetic sequence 2gcctcagcca tggtgagtct gctgcaaaca cacaaaaaga gttcaat 47 3 42 DNAartificial sequence misc_feature (1)..(42) fully synthetic sequence 3ttttttttga tatcaagctt ccatttttct tttgcataat tc 42 4 47 DNA artificialsequence misc_feature (1)..(47) fully synthetic sequence 4 gcatcggccatggtgagtct tctgcaatca aaaacataaa gatctga 47 5 40 DNA artificial sequencemisc_feature (1)..(40) fully synthetic sequence 5 ttttttttta agcttgatatcacaaccaaa tgtcaaatgg 40 6 26 DNA artificial sequence misc_feature(1)..(26) fully synthetic sequence 6 ccatctgcca tggtctatat cctgtc 26 737 DNA artificial sequence misc_feature (1)..(37) fully syntheticsequence 7 ttttttttta agcttgatat cggaagtttc tctcttg 37 8 33 DNAartificial sequence misc_feature (1)..(33) fully synthetic sequence 8cttttcccat ggtagatctc tggtcaacaa atc 33 9 1219 DNA Arabidopsis thalianapromoter (1)..(1219) Act2 promoter polynucleotide sequence and intron 9caactatttt tatgtatgca agagtcagca tatgtataat tgattcagaa tcgttttgac 60gagttcggat gtagtagtag ccattattta atgtacatac taatcgtgaa tagtgatatg 120atgaaacatt gtatcttatt gtataaatat ccataaacac atcatgaaag acactttctt 180tcacggtctg aattaattat gatacaattc taatagaaaa cgaattaaat tacgttgaat 240tgtatgaaat ctaattgaac aagccaacca cgacgacgac taacgttgcc tggattgact 300cggtttaagt taaccactaa aaaaacggag ctgtcatgta acacgcggat cgagcaggtc 360acagtcatga agccatcaaa gcaaaagaac taatccaagg gctgagatga ttaattagtt 420taaaaattag ttaacacgag ggaaaaggct gtctgacagc caggtcacgt tatctttacc 480tgtggtcgaa atgattcgtg tctgtcgatt ttaattattt ttttgaaagg ccgaaaataa 540agttgtaaga gataaacccg cctatataaa ttcatatatt ttcctctccg ctttgaattg 600tctcgttgtc ctcctcactt tcatcagccg ttttgaatct ccggcgactt gacagagaag 660aacaaggaag aagactaaga gagaaagtaa gagataatcc aggagattca ttctccgttt 720tgaatcttcc tcaatctcat cttcttccgc tctttctttc caaggtaata ggaactttct 780ggatctactt tatttgctgg atctcgatct tgttttctca atttccttga gatctggaat 840tcgtttaatt tggatctgtg aacctccact aaatcttttg gttttactag aatcgatcta 900agttgaccga tcagttagct cgattatagc taccagaatt tggcttgacc ttgatggaga 960gatccatgtt catgttacct gggaaatgat ttgtatatgt gaattgaaat ctgaactgtt 1020gaagttagat tgaatctgaa cactgtcaat gttagattga atctgaacac tgtttaagtt 1080agatgaagtt tgtgtataga ttcttcgaaa ctttaggatt tgtagtgtcg tacgttgaac 1140agaaagctat ttctgattca atcagggttt atttgactgt attgaactct ttttgtgtgt 1200ttgcagcaga ctcaccatg 1219 10 1271 DNA Arabidopsis thaliana promoter(1)..(1271) y = t/u or c Act8 promoter polynucleotide sequence andintron 10 ccatttttct tttgcataat tcatgtttat ttttttattt ttttcatcttgcataattca 60 tgtttaaaag gatatataca tgggtctact acattctcct gacattacgttttatgtgtt 120 tgtcttctga aaataatcat caaaatattt caggacttgt ttacgttttcaggagaaaaa 180 aaataactgt acccttttca atatagaaat aacatttgta gaaatcgtggattttcctta 240 ataaacaatc caaaacacga ccaccgttgt ctcctcgact cggtaacacccgatcgccga 300 cttgaaaatt agaagaaaaa tgaaaagaat aataaaaaaa aaaaaggaatgatgattgaa 360 gctgtcatat atgtcgaccc tatcacagtc aatccaatag cctatattcgccaactgata 420 tatccaacgg ctcacaaatt ttcacaaact tttcaaaaaa gtataataaaagaggctgtc 480 tgacagccat gtcacgttat actttttccg tatgatcgaa atgattcgtctttgyygaat 540 ttaattattt ccaaaattga ygactctaaa gaaaaaaaaa tagtttttcagataaacccg 600 cctatataaa tagttcaaca ctcggtttat ttcttctccc ctcaaagaattgcctcgtcg 660 tcttcagctt catcggccgt tgcatttccc ggcgataaga gagagaaagaggagaaagag 720 tgagccagtt cttcatcgtc gtggttcttg tttcttcctc gatctctcgatcttctgctt 780 ttgcttttcc gattaaggta attaaaacct ccgatctact tgttcttgtgttggatctcg 840 attacgattt ctaagttacc ttcaaaagtt gtttccgatt tgattttgattggaatttag 900 atcggtcaaa ctattggaaa tttttgatcc tggcaccgat tagctcaacgattcatgttt 960 gacttgatct tgcgttgtat ttgaaatcga tccggatcct ttcgcttcttctgtcaatag 1020 gaatctgaaa tttgaaatgt tagttgaagt ttgacttcag attctgttgatttattgact 1080 gtaacatttt gtcttccgat gagtatggat tcgttgaaat ctgctttcattatgattcta 1140 ttgatagata catcatacat tgaattgaat ctactcatga atgaaaagcctggtttgatt 1200 aagaaagtgt tttcggtttt ctcgatcaag attcagatct ttatgtttttgattgcagat 1260 cgtagaccat g 1271 11 1393 DNA Arabidopsis thalianapromoter (1)..(1393) Act11 promoter polynucleotide seqeunce and intron11 acaaccaaat gtcaaatgga atgcatcaga gaccaaacct gtaagagtcc acaaaacaat 60tcaaagaaag aatatcaaca attcagagat tcaatcctaa aacaaaaaga gaactgaaac 120caaatcgtac ctacacgacc agtgaagata ccaatagaga gctctgttgt agaatacaac 180acattaagcg caattagcag aaacagtctc ttcatctgcc gatttccact tgtcactact 240ccaaaaacct cccaaaccat ttccaaaaca gacacttttg ccatgtctac atctttccct 300tccccgaaaa acacatcatt tccatcaacg gagtaaatat ccggcggcat atcgatgctc 360gagaccgtcc tatcgagaaa aggcttagcc gcttccgtga ccgccggcgt tcgtggaccg 420tgagattgct gaaacgagcg agaataagca agcctccgat cattagcagc atatccgaca 480tcgctgctcc gatcatcagg gagctcgtta tcgcctcgag gattaaagga aatggatctc 540tccattttct tctttgatct taaagttcca acttcggcaa atactaaaat caacagtcag 600tcgtacaaag aaactctgct tatacagtaa agtcaatggg ccactgttct aagcccatat 660ataattttag aagcccatag aatacaaaag agtcaagaag cattgaccgc acaagaaaaa 720aacaattgtt aaaaagggtt ggttagtgtg tatgtatata tgaaatgcaa caaacattat 780acagcccatt aaatatggtt gttataggta gatgtcccca ttaaggaact ttatccagcc 840cattaaatta ctttacagag taaaagagag agagaagatt tacagttacg ttaccaaatt 900ttcgaaatga tttaattagt aataaataaa taattaaatg tcagttactc tctttagaaa 960gctaaataag acagctgttt ccaccaacaa cgtgactggt cgtggggtcc tccttcgttc 1020aaagtgatat tcagaaatca acggctgaga tcttctccat caatatttat tacgggccta 1080ttccttcctt ttttaaactt caattctccg gctcacattc tcttcttcat tcgctccgtt 1140tctctctcaa aaactacaca cccgtaccac accaccaccc tcctcgtttc ctcagagatc 1200ccctctctaa cttctaaggt aatcacattt ccataacgtt ccatcgtcat tgattcttca 1260ttagtatgcg tttatgaagc tttttcaatt taattctctt tggtagatct taagattcct 1320ctgtttcttg caaaataaag ggttcaatta tgctaatatt ttttatatca attttgacag 1380gatatagacc atg 1393 12 1160 DNA Arabidopsis thaliana promoter(1)..(1160) r = g or a, y = t/u or c, n = a or g or c or t/u EF1promoter polynucleotide sequence and intron 12 ggaagtttct ctcttgagggaggttgctcg tggaatggga cacatatggt tgttataata 60 aaccatttcc attgtcatgagattttgagg ttaatatata ctttacttgt tcattatttt 120 atttggtgtt tgaataaatgatataaatgg ctcttgataa tctgcattca ttgagatatc 180 aaatatttac tctagagaagagtgtcatat agattgatgg tccacaatca atgaaatttt 240 tgggagacga acatgtataaccatttgctt gaataacctt aattaaaagg tgtgattaaa 300 tgatgtttgt aacatgtagtactaaacatt cataaaacac aaccaaccca agaggtattg 360 agtattcacg gctaaacaggggcataatgg taatttaaag aatgatatta ttttatgtta 420 aaccctaaca ttggtttcggattcaacgct ataaataaaa ccactctcgt tgctgattcc 480 atttatcgtt cttattgaccctagccgcta cacacttttc tgcgatatct ctgaggtaag 540 cgttaacgta cccttaratcgttcyttttc yttttcgtct gctgatcgtt gctcatatta 600 tttcgatgat tgttggattcgatgctcttt gttgattnat cgttctgaaa attctnatct 660 gttgtttaga ttttatcgattgttaatatc aacgtttcac tgcttctaaa cgataattta 720 ttcatgaaac tattttcccattctgatcga tcttgttttg agattttaat ttgttcgatt 780 gattgttggt tggtggatctatatacgagt gaacttgttg atttgcgtat ttaagatgta 840 tgtcgatttg aattgtgattgggtaattct ggagtagcat aacaaatcca gtgttccctt 900 tttctaaggg taattctcggattgtttgct ttatatctct tgaaattgcc gatttgattg 960 aatttagctc gcttagctcagatgatagag caccacaatt tttgtggtag aaatcggttt 1020 gactccgata gcggctttttactatgattg ttttgtgtta aagatgattt tcataatggt 1080 tatatatgtc tactgtttttattgattcaa tatttgattg ttcttttttt tgcagatttg 1140 ttgaccagag atctaccatg1160 13 29 DNA artificial sequence misc_feature (1)..(29) fullysynthetic sequence 13 cccaagctta aatgacatca gatacacgc 29 14 24 DNAartificial sequence misc_feature (1)..(24) fully synthetic sequence 14cataagctta gaggtccaaa ttca 24 15 32 DNA artificial sequence misc_feature(1)..(31) fully synthetic sequence 15 ccatcagcca tggtcttcta cctttatgcaaa 32 16 36 DNA artificial sequence misc_feature (1)..(36) fullysynthetic sequence 16 ccaagcttac cacactcaga tgcataaaca aacaca 36 17 31DNA artificial sequence misc_feature (1)..(31) fully synthetic sequence17 catcagccat ggtctactct ctgcaaaaac a 31 18 27 DNA artificial sequencemisc_feature (1)..(27) fully synthetic sequence 18 gcaaagctta ctagtcaacaattggcc 27 19 28 DNA artificial sequence misc_feature (1)..(28) fullysynthetic sequence 19 gatcggccat ggttcactaa aaaaaaag 28 20 36 DNAartificial sequence misc_feature (1)..(36) fully synthetic sequence 20ggaagcttgc ggccgctttc tactctacat gtttct 36 21 31 DNA artificial sequencemisc_feature (1)..(31) fully synthetic sequence 21 gactagccgc catggttcaatctctagctg a 31 22 1578 DNA Arabidopsis thaliana promoter (1)..(1578)Act1a promoter polynucleotide sequence and intron 22 taaatgacatcagatacacg cttgtgaacc atctttaaag tattgatgga ctcttcacta 60 tgaaagctctctttaaaatt aattttcttt gtacatgtct ctaagcaatg tcaaattaat 120 tagaggtccaaattcaaaaa aatgtcgtat tgaatcattc cattactaaa ttggttcaat 180 gtcagatttaaacagcctag ggataattta gtgagatatg agattctact ttcaacatat 240 actaatcctaaatctctagc aactttttat ataagctata aatatcatga aaatgtattt 300 taatcgtttcataatttatg cagtcacact aatggaaaaa aggccaatta ttattatttt 360 cttcagactataaatgaaaa cataaattaa aatgcagatt agtttaaaat tttaataagt 420 aagtaaaatgcttatagcct tatacaaaat catatttgga agtttctaac attgttgcaa 480 tttgttatcacaaatcacag taatatttgt atactaatta gtaattacaa ctatacacaa 540 atttaaatgggtaatcatat atttgtgtcc agtggattga acaaatatgc tcggcccatg 600 cggaagtaatgccaattttg ggtgagtaaa gcccatgcga aattttcaca taagaaatgc 660 atgctttttgttttcaacga catgagttgc atgcttttta tcattgctta tatagttgca 720 agtttgcaactccttgatat tttttttatg tagacactac taccaccaaa aacttttggt 780 ctgcttattcttgtttacta tgtaaaaaaa ataaatgaat tgtttattta ctccgatttg 840 atggagtctggtttatgagg ttttatagcc tttacagaaa attgatagtt acaaaaatat 900 ttttcaaaaataaaagggta aaaccgtcat ttcaagttgt tattgttttg ggggactgga 960 tttgaaatgaaatatagaac cggaaaacaa ggtgagccga agtcgaagcc tttggacccg 1020 tttttatatttactcctccc attcccttct ccttcaatcc ttccttcctc ctcctccctt 1080 cttcttcttcccctctttca ttttccagcc actacaaact tttctatctc tacttttttt 1140 cctctcgatttcaggtactt tttgagaccc tttgttgtga ttttcgaaca cacaccccaa 1200 ttacgtttgatttttgatcc cgcatcgatt tcaattcatc cgtttctgag tttcttttgg 1260 atctgggtgtcttgagctaa tcttttcgat ctgttgttta tcgattttac tcatgcgtat 1320 gttcattacaccatttgtta tttgtttaat caaccaaaag actcatgttt ttcaaatgtc 1380 tttaatataatttttctgat tgaattttat aatatttaca tgattctgga tccagaatat 1440 ccttcttcttcttccatttt gtcctgtatt gatttgtctt tgaaaaagga ttgttctttg 1500 tatctgtattggtgaaaaag gattgttatt tgttgataaa aatttgatct ttaaacaatg 1560 tttggttttgcataaagg 1578 23 1468 DNA Arabidopsis thaliana promoter (1)..(1468)Act1b promoter polynucleotide sequence and intron 23 ttagaggtccaaattcaaaa aaatgtcgta ttgaatcatt ccattactaa attggttcaa 60 tgtcagatttaaacagccta gggataattt agtgagatat gagattctac tttcaacata 120 tactaatcctaaatctctag caacttttta tataagctat aaatatcatg aaaatgtatt 180 ttaatcgtttcataatttat gcagtcacac taatggaaaa aaggccaatt attattattt 240 tcttcagactataaatgaaa acataaatta aaatgcagat tagtttaaaa ttttaataag 300 taagtaaaatgcttatagcc ttatacaaaa tcatatttgg aagtttctaa cattgttgca 360 atttgttatcacaaatcaca gtaatatttg tatactaatt agtaattaca actatacaca 420 aatttaaatgggtaatcata tatttgtgtc cagtggattg aacaaatatg ctcggcccat 480 gcggaagtaatgccaatttt gggtgagtaa agcccatgcg aaattttcac ataagaaatg 540 catgctttttgttttcaacg acatgagttg catgcttttt atcattgctt atatagttgc 600 aagtttgcaactccttgata ttttttttat gtagacacta ctaccaccaa aaacttttgg 660 tctgcttattcttgtttact atgtaaaaaa aataaatgaa ttgtttattt actccgattt 720 gatggagtctggtttatgag gttttatagc ctttacagaa aattgatagt tacaaaaata 780 tttttcaaaaataaaagggt aaaaccgtca tttcaagttg ttattgtttt gggggactgg 840 atttgaaatgaaatatagaa ccggaaaaca aggtgagccg aagtcgaagc ctttggaccc 900 gtttttatatttactcctcc cattcccttc tccttcaatc cttccttcct cctcctccct 960 tcttcttcttcccctctttc attttccagc cactacaaac ttttctatct ctactttttt 1020 tcctctcgatttcaggtact ttttgagacc ctttgttgtg attttcgaac acacacccca 1080 attacgtttgatttttgatc ccgcatcgat ttcaattcat ccgtttctga gtttcttttg 1140 gatctgggtgtcttgagcta atcttttcga tctgttgttt atcgatttta ctcatgcgta 1200 tgttcattacaccatttgtt atttgtttaa tcaaccaaaa gactcatgtt tttcaaatgt 1260 ctttaatataatttttctga ttgaatttta taatatttac atgattctgg atccagaata 1320 tccttcttcttcttccattt tgtcctgtat tgatttgtct ttgaaaaagg attgttcttt 1380 gtatctgtattggtgaaaaa ggattgttat ttgttgataa aaatttgatc tttaaacaat 1440 gtttggttttgcataaaggt agaagacc 1468 24 1642 DNA Arabidopsis thaliana promoter(1)..(1642) Act3 promoter polynucleotide sequence and intron 24tcaagcttac cacactcaga tgcataaaca aacacagcaa gaagattgcc acaaaaatca 60taacgaaata atcaagagat agctatcaaa tcgccaccgg cgaatcatgt catactcagt 120atcagaaaca gatatgatag ctcaaaatat ggattaataa tgttactaaa cacatggaca 180ataatgcatc aatattgaaa gaaagaaaat ggtttagcag aagcaaaatg gtttagaaag 240taatgaacta cacattcaca aaggtgaaga attcgtcaag cctacaataa caaatgtcta 300tactttatga gcccacaaag agatacatca cactatctga acgaaactaa agcaacctaa 360catagtctag aaactactaa aatgaatgtt tcaaaacaat tttaacagaa ggcaaaagtg 420aaacaacata ctcctttgcg agaacgagga cgaggagcta attcacgtct ggtaacaaca 480tgtcccttgt tcaacccaac gaacaaaccg gtcttcactt gtggagttgt catcttctgt 540aaatttcata gacaacaaac aaacaaaact ttctattcaa tacaaaatca aattttacaa 600gagacggatt cagagataat aaagagatga agagagttaa atcaaaaggg attgatagaa 660gatacctaat caatggatcg agctcctccg gtggttcaga caaaagaagg acgccgactg 720aaaattacat ttttgtatat ataccagaga gactcaagaa aaaaccctag tccagtttgg 780gcttttattg ggccttataa attttgggtc agttttgaca aagtaaatac aaggctatag 840ctgctttgct aacgtgatta attatttacc atttaccaaa agccttaacc gaggccgagc 900gagaaaaaaa aacaaaaaaa aggtagaggg caagaacgtc atttccacaa ggaattgaat 960cggaaaacga ggtgtgccga attcgaagcc tttggacccg tttttatact tttttacttg 1020ccattcgttt ttttttgttc attggcctca tttgattact tgtttctttg atttctcctt 1080ccatagaacc gaattgtttt cagtctgaga tttctcctgc cgagagaacg attttaatct 1140attttcctcg gtaatgttat agcctaattt gtgttttttt ctttttcctg atccggatat 1200cgttattctg attgacaatt gtcagtttca tcttctattc tgtgaaattt tgattttttt 1260ccgatctgtg atttcgtcat tgtatcagcg tgcttatatg cgtttgaggc gtaaatgagt 1320gtgtacctca tttatcattt gctatgtttt tttttttaac agagatcttc agctgtaata 1380ttataataga ttgaattgat aacgtgattc tggatctgga atatatatat gtcacattct 1440tcttaggatt tgattttgtc tctctttgga tattaatatt cttcactccc ttgaaaatga 1500atctgtttat tataatgttt agatatattc cttaccggca tttgttttag cataaatatg 1560aaacatagca ttgactgatt tgtcttttta ttattcttgt ttttttgcca aattggtctc 1620atgtttttgc agagagtaga cc 1642 25 1241 DNA Arabidopsis thaliana promoter(1)..(1241) n= a or g or c or t/u Act7 promoter polynucleotide sequenceand intron 25 actagtcaac aattggccaa tctttngttc taaattgcta ataaacgaccatttccgtca 60 attctccttg gttgcaacag tctacccgtc aaatgtttac taatttataagtgtgaagtt 120 tgaattatga aaaacgaaat cgtattaaaa attcacaaga ataaacaactccatagattt 180 tcaaaaaaac agtcacgaga aaaaaaccac agaccgtttg tctgctcttctagtttttat 240 tatttttcta ttaatagttt tttgttattt cgagaataaa atttgaacgatgtccgaacc 300 acaaaagccg agccgataaa tcctaagccg agcctaactt tagccgtaaccatcagtcac 360 ggctcccggg ctaattcatt tgaaccgaat cataatcaac ggtttagatcaaactcaaaa 420 caatctaacg gcaacataga cgcgtcggtg agctaaaaag agtgtgaaagccaggtcacc 480 atagcattgt ctctcccaga ttttttattt gggaaataat agaagaaatagaaaaaaata 540 aaagagtgag aaaaatcgta gagctatata ttcgcacatg tactcgtttcgctttcctta 600 gtgttagctg ctgccgctgt tgtttctcct ccatttctct atctttctctctcgctgctt 660 ctcgaatctt ctgtatcatc ttcttcttct tcaaggtgag tctctagatccgttcgcttg 720 attttgctgc tcgttagtcg ttattgttga ttctctatgc cgatttcgctagatctgttt 780 agcatgcgtt gtggttttat gagaaaatct ttgttttggg ggttgcttgttatgtgattc 840 gatccgtgct tgttggatcg atctgagcta attcttaagg tttatgtgttagatctatgg 900 agtttgagga ttcttctcgc ttctgtcgat ctctcgctgt tatttttgtttttttcagtg 960 aagtgaagtt gtttagttcg aaatgacttc gtgtatgctc gattgatctggttttaatct 1020 tcgatctgtt aggtgttgat gtttacaagt gaattctagt gttttctctttgagatctgt 1080 gaagtttgaa cctagttttc tcaataatca acatatgaag cgatgtttgagtttcaataa 1140 acgctgctaa tcttcgaaac taagttgtga tctgattcgt gtttacttcatgagcttatc 1200 caattcattt cggtttcatt ttactttttt tttagtgaac c 1241 261313 DNA Arabidopsis thaliana promoter (1)..(1313) polynucleotidesequence of Act12 promoter and intron 26 tttctactct acatgtttcttgttattagg taaagtatta ggctcttttt ttaaaaaaaa 60 tgcttaatcc tctgggtacctcgaaaaggg aataatactc tagttagata agtgcagcga 120 tcaacatgac aaaatgaatgaatgtttgct ttaattggtg gctaaaagct aaatacacag 180 aaaagtcaaa attcaatctcaaaatcaacc cctctgtctc caatgtccct aatctatacc 240 aaaatgtcaa tttattttcttgatcatata ttccactaat taaaaataaa tccttctcta 300 atgaaatttg tcaaggccttggaagcctag ttttaaatat taaatggaaa ctatttcttc 360 aacaatcaca ctgttatttagtattgttgt atgttgttca ctactttctt catttgtttt 420 gtaagaaact ataataagcaaaaacacata ataaagtctc atgtcaaata atgaatctta 480 tgcacatgct tgattattttacttgcacat atccctatca tcattatcac atttgtcaat 540 taccgttatc atcattactctcattcttcc cagaactttt tcagcaattt ccatacctca 600 cccactaaga tcttttaccctttttcttaa ttatagtttg gatagcactc ttttacatag 660 cactgaaatt tcggttgaacacataaatta ctagaaacta gaaggaaatg ttactgaaat 720 ttcactgatt gtctaaaattgaataatcta aagaaaatgg ccttttaacc tttttcttag 780 gcccaaatgg gctcattaccactcatgctt gttcggtgac ccgattcttc cggtaaaaca 840 gagcctaaac cgtattttcaggttaggctg gtgttttctt aattctccaa cctaaaaata 900 gatggacacg tgtctatagaggctgagata ttggtctcaa tgaagaaaac taacggctca 960 gacccgtgta tgaacgatattaagggccaa agttgcttct gttttccaga aatttttgaa 1020 acccaatttc agggcacgattccacaacct ctttcttttc ttctagatct acgtaaattc 1080 atcaggtaca tgttattttttttgtttatt tgatgtcaaa attttgatca caaggaggca 1140 aaaccaatat aaatgtaacgctaatgcgtt tgattatggt atacgtaacg aattagattt 1200 aatggttaca ttttattgttttagatttag ttatgagatt ggcattaatt attggtgttt 1260 cctttgaatt tgctatgtttcttatgttga tgtaatcagc tagagattga acc 1313 27 1946 DNA artificialsequence promoter (1)..(1946) chimeric promoter fusion FMV and Act11polynucleotides + Act11 intro 27 aattctcagt ccaaagcctc aacaaggtcagggtacagag tctccaaacc attagccaaa 60 agctacagga gatcaatgaa gaatcttcaatcaaagtaaa ctactgttcc agcacatgca 120 tcatggtcag taagtttcag aaaaagacatccaccgaaga cttaaagtta gtgggcatct 180 ttgaaagtaa tcttgtcaac atcgagcagctggcttgtgg ggaccagaca aaaaaggaat 240 ggtgcagaat tgttaggcgc acctaccaaaagcatctttg cctttattgc aaagataaag 300 cagattcctc tagtacaagt ggggaacaaaataacgtgga aaagagctgt cctgacagcc 360 cactcactaa tgcgtatgac gaacgcagtgacgaccacaa aagaattagc ttgagctcag 420 gatttagcag cattccagat tgggttcaatcaacaaggta cgagccatat cactttattc 480 aaattggtat cgccaaaacc aagaaggaactcccatcctc aaaggtttgt aaggaagaat 540 tcgatatccc cgcggccgcg ttatcacaaccaaatgtcaa atggaatgca tcagagacca 600 aacctgtaag agtccacaaa acaattcaaagaaagaatat caacaattca gagattcaat 660 cctaaaacaa aaagagaact gaaaccaaatcgtacctaca cgaccagtga agataccaat 720 agagagctct gttgtagaat acaacacattaagcgcaatt agcagaaaca gtctcttcat 780 ctgccgattt ccacttgtca ctactccaaaaacctcccaa accatttcca aaacagacac 840 ttttgccatg tctacatctt tcccttccccgaaaaacaca tcatttccat caacggagta 900 aatatccggc ggcatatcga tgctcgagaccgtcctatcg agaaaaggct tagccgcttc 960 cgtgaccgcc ggcgttcgtg gaccgtgagattgctgaaac gagcgagaat aagcaagcct 1020 ccgatcatta gcagcatatc cgacatcgctgctccgatca tcagggagct cgttatcgcc 1080 tcgaggatta aaggaaatgg atctctccattttcttcttt gatcttaaag ttccaacttc 1140 ggcaaatact aaaatcaaca gtcagtcgtacaaagaaact ctgcttatac agtaaagtca 1200 atgggccact gttctaagcc catatataattttagaagcc catagaatac aaaagagtca 1260 agaagcattg accgcacaag aaaaaaacaattgttaaaaa gggttggtta gtgtgtatgt 1320 atatatgaaa tgcaacaaac attatacagcccattaaata tggttgttat aggtagatgt 1380 ccccattaag gaactttatc cagcccattaaattacttta cagagtaaaa gagagagaga 1440 agatttacag ttacgttacc aaattttcgaaatgatttaa ttagtaataa ataaataatt 1500 aaatgtcagt tactctcttt agaaagctaaataagacagc tgtttccacc aacaacgtga 1560 ctggtcgtgg ggtcctcctt cgttcaaagtgatattcaga aatcaacggc tgagatcttc 1620 tccatcaata tttattacgg gcctattccttcctttttta aacttcaatt ctccggctca 1680 cattctcttc ttcattcgct ccgtttctctctcaaaaact acacacccgt accacaccac 1740 caccctcctc gtttcctcag agatcccctctctaacttct aaggtaatca catttccata 1800 acgttccatc gtcattgatt cttcattagtatgcgtttat gaagcttttt caatttaatt 1860 ctctttggta gatcttaaga ttcctctgtttcttgcaaaa taaagggttc aattatgcta 1920 atatttttta tatcaatttt gacagg 194628 1695 DNA artificial sequence promoter (1)..(1695) n= a or g or c ort/u, r = g or a, y = t/u or c chimeric promoter fusion FMV and EF1polynucleotides + EF1 intro 28 aattctcagt ccaaagcctc aacaaggtcagggtacagag tctccaaacc attagccaaa 60 agctacagga gatcaatgaa gaatcttcaatcaaagtaaa ctactgttcc agcacatgca 120 tcatggtcag taagtttcag aaaaagacatccaccgaaga cttaaagtta gtgggcatct 180 ttgaaagtaa tcttgtcaac atcgagcagctggcttgtgg ggaccagaca aaaaaggaat 240 ggtgcagaat tgttaggcgc acctaccaaaagcatctttg cctttattgc aaagataaag 300 cagattcctc tagtacaagt ggggaacaaaataacgtgga aaagagctgt cctgacagcc 360 cactcactaa tgcgtatgac gaacgcagtgacgaccacaa aagaattagc ttgagctcag 420 gatttagcag cattccagat tgggttcaatcaacaaggta cgagccatat cactttattc 480 aaattggtat cgccaaaacc aagaaggaactcccatcctc aaaggtttgt aaggaagaat 540 tcgatatcaa gcttgatatc ggaagtttctctcttgaggg aggttgctcg tggaatggga 600 cacatatggt tgttataata aaccatttccattgtcatga gattttgagg ttaatatata 660 ctttacttgt tcattatttt atttggtgtttgaataaatg atataaatgg ctcttgataa 720 tctgcattca ttgagatatc aaatatttactctagagaag agtgtcatat agattgatgg 780 tccacaatca atgaaatttt tgggagacgaacatgtataa ccatttgctt gaataacctt 840 aattaaaagg tgtgattaaa tgatgtttgtaacatgtagt actaaacatt cataaaacac 900 aaccaaccca agaggtattg agtattcacggctaaacagg ggcataatgg taatttaaag 960 aatgatatta ttttatgtta aaccctaacattggtttcgg attcaacgct ataaataaaa 1020 ccactctcgt tgctgattcc atttatcgttcttattgacc ctagccgcta cacacttttc 1080 tgcgatatct ctgaggtaag cgttaacgtacccttaratc gttcyttttc yttttcgtct 1140 gctgatcgtt gctcatatta tttcgatgattgttggattc gatgctcttt gttgattnat 1200 cgttctgaaa attctnatct gttgtttagattttatcgat tgttaatatc aacgtttcac 1260 tgcttctaaa cgataattta ttcatgaaactattttccca ttctgatcga tcttgttttg 1320 agattttaat ttgttcgatt gattgttggttggtggatct atatacgagt gaacttgttg 1380 atttgcgtat ttaagatgta tgtcgatttgaattgtgatt gggtaattct ggagtagcat 1440 aacaaatcca gtgttccctt tttctaagggtaattctcgg attgtttgct ttatatctct 1500 tgaaattgcc gatttgattg aatttagctcgcttagctca gatgatagag caccacaatt 1560 tttgtggtag aaatcggttt gactccgatagcggcttttt actatgattg ttttgtgtta 1620 aagatgattt tcataatggt tatatatgtctactgttttt attgattcaa tatttgattg 1680 ttcttttttt tgcag 1695 29 1800 DNAartificial sequence promoter (1)..(1800) y = t/u or c chimeric promoterfusion CaMV and Act8 polynucleotides + Act8 intro 29 ggtccgatgtgagacttttc aacaaagggt aatatccgga aacctcctcg gattccattg 60 cccagctatctgtcacttta ttgtgaagat agtggaaaag gaaggtggct cctacaaatg 120 ccatcattgcgataaaggaa aggccatcgt tgaagatgcc tctgccgaca gtggtcccaa 180 agatggacccccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc 240 aaagcaagtggattgatgtg atggtccgat gtgagacttt tcaacaaagg gtaatatccg 300 gaaacctcctcggattccat tgcccagcta tctgtcactt tattgtgaag atagtggaaa 360 aggaaggtggctcctacaaa tgccatcatt gcgataaagg aaaggccatc gttgaagatg 420 cctctgccgacagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag 480 aagacgttccaaccacgtct tcaaagcaag tggattgatg tgatatcaag cttccatttt 540 tcttttgcataattcatgtt tattttttta tttttttcat cttgcataat tcatgtttaa 600 aaggatatatacatgggtct actacattct cctgacatta cgttttatgt gtttgtcttc 660 tgaaaataatcatcaaaata tttcaggact tgtttacgtt ttcaggagaa aaaaaataac 720 tgtacccttttcaatataga aataacattt gtagaaatcg tggattttcc ttaataaaca 780 atccaaaacacgaccaccgt tgtctcctcg actcggtaac acccgatcgc cgacttgaaa 840 attagaagaaaaatgaaaag aataataaaa aaaaaaaagg aatgatgatt gaagctgtca 900 tatatgtcgaccctatcaca gtcaatccaa tagcctatat tcgccaactg atatatccaa 960 cggctcacaaattttcacaa acttttcaaa aaagtataat aaaagaggct gtctgacagc 1020 catgtcacgttatacttttt ccgtatgatc gaaatgattc gtctttgyyg aatttaatta 1080 tttccaaaattgaygactct aaagaaaaaa aaatagtttt tcagataaac ccgcctatat 1140 aaatagttcaacactcggtt tatttcttct cccctcaaag aattgcctcg tcgtcttcag 1200 cttcatcggccgttgcattt cccggcgata agagagagaa agaggagaaa gagtgagcca 1260 gatcttcatcgtcgtggttc ttgtttcttc ctcgatctct cgatcttctg cttttgcttt 1320 tccgattaaggtaattaaaa cctccgatct acttgttctt gtgttggatc tcgattacga 1380 tttctaagttaccttcaaaa gttgtttccg atttgatttt gattggaatt tagatcggtc 1440 aaactattggaaatttttga tcctggcacc gattagctca acgattcatg tttgacttga 1500 tcttgcgttgtatttgaaat cgatccggat cctttcgctt cttctgtcaa taggaatctg 1560 aaatttgaaatgttagttga agtttgactt cagattctgt tgatttattg actgtaacat 1620 tttgtcttccgatgagtatg gattcgttga aatctgcttt cattatgatt ctattgatag 1680 atacatcatacattgaattg aatctactca tgaatgaaaa gcctggtttg attaagaaag 1740 tgttttcggttttctcgatc aagattcaga tctttatgtt tttgattgca gatcgtagac 1800 30 1742 DNAartificial sequence promoter (1)..(1742) chimeric promoter fusion CaMVand Act2 polynucleotides + Act2 intro 30 gtccgatgtg agacttttcaacaaagggta atatccggaa acctcctcgg attccattgc 60 ccagctatct gtcactttattgtgaagata gtggaaaagg aaggtggctc ctacaaatgc 120 catcattgcg ataaaggaaaggccatcgtt gaagatgcct ctgccgacag tggtcccaaa 180 gatggacccc cacccacgaggagcatcgtg gaaaaagaag acgttccaac cacgtcttca 240 aagcaagtgg attgatgtgatggtccgatg tgagactttt caacaaaggg taatatccgg 300 aaacctcctc ggattccattgcccagctat ctgtcacttt attgtgaaga tagtggaaaa 360 ggaaggtggc tcctacaaatgccatcattg cgataaagga aaggccatcg ttgaagatgc 420 ctctgccgac agtggtcccaaagatggacc cccacccacg aggagcatcg tggaaaaaga 480 agacgttcca accacgtcttcaaagcaagt ggattgatgt gatatcaagc ttcaactatt 540 tttatgtatg caagagtcagcatatgtata attgattcag aatcgttttg acgagttcgg 600 atgtagtagt agccattatttaatgtacat actaatcgtg aatagtgata tgatgaaaca 660 ttgtatctta ttgtataaatatccataaac acatcatgaa agacactttc tttcacggtc 720 tgaattaatt atgatacaattctaatagaa aacgaattaa attacgttga attgtatgaa 780 atctaattga acaagccaaccacgacgacg actaacgttg cctggattga ctcggtttaa 840 gttaaccact aaaaaaacggagctgtcatg taacacgcgg atcgagcagg tcacagtcat 900 gaagccatca aagcaaaagaactaatccaa gggctgagat gattaattag tttaaaaatt 960 agttaacacg agggaaaaggctgtctgaca gccaggtcac gttatcttta cctgtggtcg 1020 aaatgattcg tgtctgtcgattttaattat ttttttgaaa ggccgaaaat aaagttgtaa 1080 gagataaacc cgcctatataaattcatata ttttcctctc cgctttgaat tgtctcgttg 1140 tcctcctcac tttcatcagccgttttgaat ctccggcgac ttgacagaga agaacaagga 1200 agaagactaa gagagaaagtaagagataat ccaggagatt cattctccgt tttgaatctt 1260 cctcaatctc atcttcttccgctctttctt tccaaggtaa taggaacttt ctggatctac 1320 tttatttgct ggatctcgatcttgttttct caatttcctt gagatctgga attcgtttaa 1380 tttggatctg tgaacctccactaaatcttt tggttttact agaatcgatc taagttgacc 1440 gatcagttag ctcgattatagctaccagaa tttggcttga ccttgatgga gagatccatg 1500 ttcatgttac ctgggaaatgatttgtatat gtgaattgaa atctgaactg ttgaagttag 1560 attgaatctg aacactgtcaatgttagatt gaatctgaac actgtttaag ttagatgaag 1620 tttgtgtata gattcttcgaaactttagga tttgtagtgt cgtacgttga acagaaagct 1680 atttctgatt caatcagggtttatttgact gtattgaact ctttttgtgt gtttgcagca 1740 ga 1742

We claim:
 1. A DNA construct comprising: an expression cassettecomprising a promoter DNA sequence comprising at least one cis elementderived from a sequence selected from the group consisting of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26; a structural DNAsequence that encodes an agronomically useful protein; and a 3′non-translated region that functions in plants to cause the addition ofpolyadenylated nucleotides to the 3′ end of the RNA sequence; whereinthe structural gene is operably linked to the promoter and the 3′non-translated region, and the promoter. DNA sequence is heterologouswith respect to the structural DNA sequence.
 2. A DNA construct of claim1, wherein the structural DNA sequence encodes a herbicide tolerancegene.
 3. A DNA construct of claim 2, wherein the herbicide tolerancegene is a glyphosate tolerance gene.
 4. A DNA construct of claim 3,wherein the glyphosate tolerance gene is an EPSP synthase gene or aglyphosate oxidoreductase gene.
 5. A DNA construct of claim 4, whereinthe glyphosate tolerance gene is an aroA:CP4 gene.
 6. A DNA construct ofclaim 3, that, when introduced into a plant cell, confers to the plantcell tolerance to an aqueous glyphosate formulation comprising at least50 grams acid equivalent per liter of glyphosate.
 7. A DNA construct ofclaim 6, that, when introduced into a plant cell, confers to the plantcell tolerance to an aqueous glyphosate formulation comprising at least300 grams acid equivalent per liter of glyphosate.
 8. A DNA construct ofclaim 3, that, when introduced into a plant cell, confers to the plantcell tolerance to at least one application of glyphosate at a rate of 16oz per acre.
 9. A DNA construct of claim 1, wherein the structural DNAsequence is a Bacillus thuringiensis insect control gene.
 10. A DNAconstruct of claim 3, wherein the promoter DNA sequence consistsessentially of a 5′ regulatory region derived from a sequence selectedfrom the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, and SEQ ID NO:
 26. 11. A chimeric promoter DNA sequence comprisingat least one cis element derived from a sequence selected from the groupconsisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26;and said at least one cis element is operably linked to a heterologouspromoter sequence.
 12. A chimeric promoter DNA sequence of claim 11,wherein the heterologous promoter sequence is a plant DNA viruspromoter.
 13. A chimeric promoter DNA sequence of claim 12 wherein theplant DNA virus promoter is selected from the group consisting ofCauliflower mosaic virus and Figwort mosaic virus.
 14. A chimericpromoter DNA sequence of claim 13, wherein the chimeric promoter DNAsequence is selected from the group consisting of SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, and SEQ ID NO:30.
 15. A DNA construct comprising:and expression cassette comprising a chimeric promoter DNA sequencecomprising at least one cis element derived from a sequence selectedfrom the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26; and said at least one cis element is operably linked to aheterologous promoter sequence; a structural DNA sequence; and a 3′non-translated region that functions in plants to cause the addition ofpolyadenylated nucleotides to the 3′ end of the RNA sequence; whereinthe structural gene is operably linked to the promoter and the 3′non-translated region, and the promoter DNA sequence is heterologouswith respect to the structural DNA sequence.
 16. A DNA construct ofclaim 15, wherein the structural DNA sequence encodes a herbicidetolerance gene.
 17. A DNA construct of claim 16, wherein the herbicidetolerance gene is a glyphosate tolerance gene.
 18. A DNA construct ofclaim 17, wherein the glyphosate tolerance gene is an EPSP synthase geneor a glyphosate oxidoreductase gene.
 19. A DNA construct of claim 17,wherein the glyphosate tolerance gene is an aroA:CP4 gene.
 20. A DNAconstruct of claim 17, that, when introduced into a plant cell, confersto the plant cell tolerance to an aqueous glyphosate formulationcomprising at least 50 grams acid equivalent per liter of glyphosate.21. A DNA construct of claim 17, that, when introduced into a plantcell, confers to the plant cell tolerance to an aqueous glyphosateformulation comprising at least 300 grams acid equivalent per liter ofglyphosate.
 22. A DNA construct of claim 17, that, when introduced intoa plant cell, confers to the plant cell tolerance to at least oneapplication of glyphosate at a rate of 16 oz per acre.
 23. A DNAconstruct of claim 15, wherein the structural DNA sequence is an insectcontrol gene.
 24. A DNA construct of claim 23, wherein the insectcontrol gene is derived from Bacillus thuringieneis.
 25. A DNA constructcomprising a chimeric promoter DNA sequence selected from the groupconsisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ IDNO:30; a structural DNA sequence; and a 3′ non-translated region thatfunctions in plants to cause the addition of polyadenylated nucleotidesto the 3′ end of the RNA sequence; wherein the structural gene isoperably linked to the chimeric promoter and the 3′ non-translatedregion, and the chimeric promoter DNA sequence is heterologous withrespect to the structural DNA sequence.
 26. A DNA construct of claim 25,wherein the structural DNA sequence encodes a herbicide tolerance gene.27. A DNA construct of claim 26, wherein the herbicide tolerance gene isa glyphosate tolerance gene.
 28. A DNA construct of claim 27, whereinthe glyphosate tolerance gene is an EPSP synthase gene or a glyphosateoxidoreductase gene.
 29. A DNA construct of claim 27, wherein theglyphosate tolerance gene is an aroA:CP4 gene.
 30. A DNA construct ofclaim 27, that, when introduced into a plant cell, confers to the plantcell tolerance to an aqueous glyphosate formulation comprising at least50 grams acid equivalent per liter of glyphosate.
 31. A DNA construct ofclaim 27, that, when introduced into a plant cell, confers to the plantcell tolerance to an aqueous glyphosate formulation comprising at least300 grams acid equivalent per liter of glyphosate.
 32. A DNA constructof claim 27, that, when introduced into a plant cell, confers to theplant cell tolerance to at least one application of glyphosateformulation at a rate of 16 oz per acre.
 33. A transgenic crop plantcomprising the DNA construct of claim
 2. 34. A transgenic crop plant ofclaim 33, wherein said crop plant is a monocot crop species.
 35. Atransgenic crop plant of claim 33, wherein said crop plant is a dicotcrop species.
 36. A transgenic crop plant of claim 33, wherein theherbicide tolerance gene is a glyphosate tolerance gene.
 37. Atransgenic crop plant comprising the DNA construct of claim
 9. 38. Atransgenic crop plant of claim 37, wherein said crop plant is a monocotcrop species.
 39. A transgenic crop plant of claim 37, wherein said cropplant is a dicot crop species.
 40. A transgenic crop plant comprisingthe DNA construct of claim
 15. 41. A transgenic crop plant of claim 40,wherein said crop plant is a monocot crop species.
 42. A transgenic cropplant of claim 40, wherein said crop plant is a dicot crop species. 43.A transgenic crop plant comprising the DNA construct of claim
 25. 44. Atransgenic crop plant of claim 43, wherein said crop plant is a monocotcrop species.
 45. A transgenic crop plant of claim 43, wherein said cropplant is a dicot crop species.
 46. A DNA construct comprising a firstexpression cassette and a second expression cassette, wherein said firstexpression cassette comprises a promoter DNA sequence comprising atleast one cis element derived from a sequence selected from the groupconsisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26; astructural DNA sequence that encodes an agronomically useful protein;and a 3′ non-translated region that functions in plants to cause theaddition of polyadenylated nucleotides to the 3′ end of the RNAsequence; wherein the structural gene is operably linked to the promoterand the 3′ non-translated region, and the promoter DNA sequence isheterologous with respect to the structural DNA sequence; and saidsecond expression cassette comprises a promoter DNA sequence comprisingat least one cis element derived from a sequence selected from the groupconsisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26; astructural DNA sequence that encodes an agronomically useful protein;and a 3′ non-translated region that functions in plants to cause theaddition of polyadenylated nucleotides to the 3′ end of the RNAsequence; wherein the structural gene is operably linked to the promoterand the 3′ non-translated region, and the promoter DNA sequence isheterologous with respect to the structural DNA sequence.
 47. A methodof expressing a structural DNA sequence in a plant, the methodcomprising: (1) providing a DNA construct comprising a promoter that isfunctional in a plant cell, the promoter comprising at least one ciselement derived from a sequence selected from the group consisting ofSEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12;, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26; astructural DNA sequence encoding an agronomically useful protein; and a3′ non-translated region that functions to cause the addition ofpolyadenylated nucleotides to the 3′ end of the RNA sequence; whereinthe structural gene is operably linked to the promoter and the 3′non-translated region, and the promoter is heterologous with respect tothe structural DNA sequence; (2) introducing the DNA construct into aplant cell; and (3) regenerating the plant cell to produce the plantsuch that the structural DNA sequence is expressible in the plant.
 48. Amethod of expressing a structural DNA sequence in a plant, the methodcomprising: (1) providing a DNA construct comprising a promoter that isfunctional in a plant cell, the promoter comprising a sequence selectedfrom the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,and SEQ ID NO:30; a structural DNA sequence; and a 3′ non-translatedregion that functions to cause the addition of polyadenylatednucleotides to the 3′ end of the RNA sequence; wherein the structuralgene is operably linked to the promoter and the 3′ non-translatedregion, and the promoter is heterologous with respect to the structuralDNA sequence; (2) introducing the DNA construct into a plant cell; and(3) regenerating the plant cell to produce the plant such that thestructural DNA sequence is expressible in the plant.
 49. A method ofcontrolling weeds, the method comprising: (1) providing a crop planttransformed with a DNA construct that comprises a promoter that isfunctional in a plant cell, the promoter comprising at least one ciselement derived from a sequence selected from the group consisting ofSEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12;, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26; aglyphosate tolerance gene; and a 3′ non-translated region that functionsto cause the addition of polyadenylated nucleotides to the 3′ end of theRNA sequence; wherein the glyphosate tolerance gene is operably linkedto the promoter and the 3′ non-translated region, and the promoter isheterologous with respect to the glyphosate tolerance gene; and (2)applying to the crop plant a sufficient amount of glyphosate to controlweeds without damaging the crop plant.
 50. A method of controllingweeds, the method comprising: (1) providing a crop plant transformedwith a DNA construct that comprises a promoter that is functional in aplant cell, the promoter comprising a sequence selected from the groupconsisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29; and SEQ IDNO:30; a glyphosate tolerance gene; and a 3′ non-translated region thatfunctions to cause the addition of polyadenylated nucleotides to the 3′end of the RNA sequence; wherein the glyphosate tolerance gene isoperably linked to the promoter and the 3′ non-translated region, andthe promoter is heterologous with respect to the glyphosate tolerancegene; and (2) applying to the crop plant a sufficient amount ofglyphosate to control weeds without damaging the crop plant.